Preparation of antibiotic cryptdin peptides

ABSTRACT

The present invention provides a method of preparing substantially purified cryptdin peptides having a consensus amino acid sequence: 
     
         X.sub.1 -C-X.sub.2 -C-R-X.sub.3 -C-X.sub.4 -E-X.sub.5 -C-X.sub.6 
    
      -C-C-X 7   
     wherein X 1  is 3 to 9 amino acids; X 2  is one amino acid, preferably Y, H or R; X 3  is 2 or 3 amino acids; X 4  is three amino acids; X 5  is five amino acids; X 6  is 6 to 10 amino acids; and X 7  is 0 to 9 amino acids. The invention also provides a method of preparing substantially purified mouse cryptdin having a consensus amino acid sequence: 
     
         X.sub.1 -L-X.sub.2 -C-Y-C-R-X.sub.3 -C-K-X.sub.4 -E-X.sub.5 -G-T-C-X.sub.6 
    
      -C-C-X 7   
     wherein X 1  is 3 or 4 amino acids, preferably LRD, LSKK (SEQ ID NO: 1) or LRG; X 2  is 1 amino acid, preferably V, L or I; X 3  is 3 amino acids, preferably KGH or *RG, where * is S, T, K, I or A; X 4  is 2 amino acids, preferably GR, RR or RG; X 5  is 3 amino acids, preferably RMN, RVR, RVF HMN or HIN; X 6  is 6 to 9 amino acids, preferably GIRFLY (SEQ ID NO: 2) or RNLFLTFVF (SEQ ID NO: 3), RRGHLMYTL (SEQ ID NO: 4) or RKGHL*YT* (SEQ ID NO: 5), where * independently is L or M; and X 7  is 0 to 3 amino acids, preferably R, S or PRR. The invention also provides a method of preparing cryptdin analogs, which are devoid of one or more amino acids N-terminal to the first cysteine.

This invention was made with government support under grant numbersAI22931, AI31696, DK08851, DK44632 and DK33506, awarded by NationalInstitutes of Health. The Government has certain rights in theinvention.

This application is a continuation of U.S. Ser. No. 08/482,279, filedJun. 7, 1995, now U.S. Pat. No. 5,840,498, issued Nov. 24, 1998; whichis a continuation of U.S. Ser. No. 08/342,268, filed Nov. 18, 1994, nowU.S. Pat. No. 5,844,072, issued Dec. 1, 1998; which is acontinuation-in-part of U.S. Ser. No. 07/930,649, filed Aug. 14, 1992,now U.S. Pat. No. 5,422,424, issued Jun. 6, 1995; which is acontinuation-in-part of U.S. Ser. No. 07/889,020, filed May 26, 1992,now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to antimicrobial peptides and morespecifically to cryptdin peptides, nucleic acid molecules encodingcryptdins, and their uses.

2. Background Information

Survival in a world teaming with microorganisms depends on a network ofhost defense mechanisms. Among these mechanisms are phagocytosis bycells are resident in tissues or that circulate in the blood system andingest, kill and digest potentially harmful microbes. Althoughpathogenic microbes may vary considerably, phagocytes are able todestroy the vast majority by sequestering them in intracytoplasmicvacuoles and exposing them to a lethal mixture of organic and inorganictoxins.

Perhaps the most remarkable ultrastructural feature of phagocytes aretheir several thousand cytoplasmic granules, which are membrane-boundorganelles typically about 0.3 μm in diameter. During phagocytosis, someof these granules fuse to phagocytic vesicles thus enabling the contentsof the granule to enter the lumen of the vesicle. Early observerssurmised correctly that the granules contained factors which wereresponsible for intraphagosomal killing in digestion of microbes. Thesegranules contain a mixture of antimicrobial molecules including variouspeptides such as the so-called defensins.

Defensins are abundant antimicrobial peptide components of vertebrateneutrophil and macrophage granules. Members of the defensin family havebeen identified previously in human, rabbit, guinea pig and ratphagocytes, primarily those phagocytes termed phagocytic granulocytes.Defensins are cationic peptides that have molecular weights betweenabout 3 and 4 kiloDaltons (kDa) and that exhibit broad-rangeantimicrobial activities against gram negative and gram positivebacteria, many fungi and some enveloped viruses. The peptides arecharacterized by eight invariant amino acids, including six invariantcysteine residues that constitute a unique disulfide motif. The threedisulfide bonds stabilize a tertiary conformation consistingpredominantly of β-sheet. The highly ordered structure and the absenceof a helix make defensins unique among known antimicrobial peptides. Itappears that defensins exert their antibacterial effect bypermeabilizing the cytoplasmic membrane of the target microorganism by amechanism that may involve the formation of ion channels ortransmembrane pores.

Until recently, defensins had been identified only in circulating ortissue phagocytes of myeloid origin. However, based on the presence of aparticular mRNA, it has been surmised that similar peptides might bepresent in the epithelial cells of the small intestine. Such intestinalpeptides may prevent access of microorganisms through the smallintestine into the systemic circulation and, therefore, can be useful asa therapeutic or prophylactic agent. Thus, a need exists to identifypeptides that have antimicrobial activity within the mucosal epitheliumor in the intestinal lumen. The present invention satisfies this needand provides additional benefits as well.

SUMMARY OF THE INVENTION

The present invention provides a substantially purified cryptdin peptidehaving a consensus amino acid sequence:

    X.sub.1 -C-X.sub.2 -C-R-X.sub.3 -C-X.sub.4 -E-X.sub.5 -C-X.sub.6 -C-C-X.sub.7

wherein X₁ is 3 to 9 amino acids; X₂ is 1 amino acid, preferably Y, H orR; X₃ is 2 or 3 amino acids; X₄ is 3 amino acids; X₅ is 5 amino acids;X₆ is 6 to 10 amino acids; and X₇ is 0 to 9 amino acids.

The invention also provides a substantially purified mouse cryptdinhaving a consensus amino acid sequence:

    X.sub.1 -L-X.sub.2 -C-Y-C-R-X.sub.3 -C-K-X.sub.4 -E-X.sub.5 -G-T-C-X.sub.6 -C-C-X.sub.7

wherein X₁ is 3 or 4 amino acids, preferably LRD, LSKK (SEQ ID NO: 1) orLRG;

X₂ is 1 amino acid, preferably V, L or I;

X₃ is 3 amino acids, preferably KGH or *RG, where * is S, T, K, I or A;

X₄ is 2 amino acids, preferably GR, RR or RG;

X₅ is 3 amino acids, preferably RMN, RVR, RVF HMN or HIN;

X₆ is 6 to 9 amino acids, preferably GIRFLY (SEQ ID NO: 2) or RNLFLTFVF(SEQ ID NO: 3), RRGHLMYTL (SEQ ID NO: 4) or RKGHL*YT* (SEQ ID NO: 5),where * independently is L or M; and

X₇ is 0 to 3 amino acids, preferably R, S or PRR.

For example, the invention provides various mouse, rat or humancryptdins having the sequence:

    __________________________________________________________________________     1)                                                                             LRDLVCYCRSRGCKGRERMNGTCRKGHLLYTLCCR                                                                          (SEQ ID NO: 6);                                 2) LRDLVCYCRTRGCKRRERMNGTCRKGHLMYTLCCR (SEQ ID NO: 7);                        3) LRDLVCYCRKRGCKRRERMNGTCRKGHLMYTLCCR (SEQ ID NO: 8);                        4) GLLCYCRKGHCKRGERVRGTCGIRFLYCCPR (SEQ ID NO: 9);                            5) LSKKLICYCRIRGCKRRERVFGTCRNLFLTFVFCC (SEQ ID NO: 10);                       6) LKQCHCRKFCRPYEKAEGSCRPGLFIKRKICCIQQWTPG (SEQ ID NO: 11);                   7) GLLCYCRKGHCKRGERVRGTCGIRFLYCCPRR (SEQ ID NO: 12);                          8) LSKKLICYCRIRGCKRRERVFGTCRNLFLTFVFCCS (SEQ ID NO: 13);                      9) LRDLVCYCRARGCKGRERMNGTCRKGHLLYMLCCR (SEQ ID NO: 14);                      10) LKQCHCRKFCRPYEKAEGSCRPGLFIKRKICCIQQWTPGRT (SEQ ID NO: 15);                11) IGRPVRRCRCRANCGPKEYATAFCAQGPFKQFKFCCT (SEQ ID NO: 16);                    12) IRWPWKRCHCRSFCRPYENATSFCAQGLFKQHKFCCLDTWPPRMK (SEQ ID NO: 17);                                            13) TSGSQARATCYCRTGRCATRESLSGVCEISGRLYRL                                     CCR (SEQ ID NO: 18); and                       14) AFTCHCRRSCYSTEYSYGTCTVMGINHRFCCL (SEQ ID NO: 19).                       __________________________________________________________________________

Cryptdins are typically characterized by being naturally found in theepithelial cells of the small intestine, being cationic, being about 30to about 45 amino acids in length, having at least three and,preferably, three to nine, amino acids to the N-terminal of the firstcysteine residue, exhibiting specific antimicrobial activity againstintestinal pathogens and opportunistic pathogens and being relativelynon-toxic to cells of the host organism. However, there may be diversityin these structural and functional characteristics. The invention alsoprovides cryptdin analogs, which are devoid of one or more amino acidsN-terminal to the first cysteine. In addition, the invention alsoprovides nucleic acid molecules encoding cryptdin peptides. For example,the invention provides genomic DNA sequences and cDNA sequences encodingmouse and rat cryptdins.

The invention further provides a method for detecting an inflammatorypathology in a subject by determining the amount of cryptdin in abiological sample from the subject and comparing that amount to theamount present in a normal subject. Such a method can be used todetermine the presence of an inflammatory pathology such as inflammatorybowel disease, pancreatitis, malignancy, infection or ileitis.

The invention also provides a method for treating an inflammatorypathology in a subject by administering a cryptdin to the subject. Suchtreatment is particularly advantageous in patients who areimmunocompromised due, for example, to malnutrition, radiation burns,immunosuppressive infections, autoimmune disease, neonatality, bonemarrow transplantation or chemotherapy. A cryptdin can be administeredorally, by nasogastric intubation, by transabdominal catheter,intravenously or by aerosol inhalation. When administered orally, it ispreferably in a delayed release formulation designed to permit releasein the small intestine. The cryptdin can be administered as acomposition with a physiologically acceptable medium, and more than onecryptdin can be administered simultaneously or sequentially.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the structures of mouse cryptdins 1-5 (SEQ ID NOS: 6 to10, respectively) and rat cryptdin 1 (SEQ ID NO: 11). Amino acidresidues are indicated by single letter code. Dashed lines are includedin mouse cryptdin 4 (SEQ ID NO: 9) and rat cryptdin 1 (SEQ ID NO: 11) inorder to preserve the consensus sequence where these peptides areshorter than other cryptdins. Invariant residues in the enteric cryptdinpeptides are boxed. Disulfide bonding motifs are depicted by connectingdouble lines.

FIGS. 2.A. to 2.C. show chromatograms representing the purification ofenteric cryptdins. Acid extract of jejunum and ileum was chromatographedin 30% acetic acid on a P-60 column. Fractions indicated by the bracket(FIG. 2.A.) were pooled and rechromatographed on the P-60 column (FIG.2.B.). Cryptdin containing fractions (bracket, panel B) were pooled andfurther purified by reversed-phase high performance liquidchromatography (RP-HPLC) on 0.46×25 cm Vydac C-18 column.Water-acetonitrile gradient elution (--) using 0.13% (vol/vol) HFBA asmodifier was used to purify cryptdins 1-5 (SEQ ID NOS: 6 to 10,respectively). The brackets in FIG. 2.C. indicate the peptide containedin each peak, and the portion of each which was subjected to a secondround of RP-HPLC.

FIG. 3 shows acid-urea PAGE of purified enteric cryptdins. Samples oflow molecular weight enteric peptides obtained by P-60 gel filtration(FIG. 2, panel B) and purified cryptdins were electrophoresed on a 12.5%acid-urea gel and stained with formalin-containing Coomassie Blue. LaneA: approximately 20 μg P-60 low molecular weight peptide fractions;lanes B-F: 1 μg each of cryptdins 1-5 (SEQ ID NOS: 6 to 10),respectively.

FIGS. 4.A. and 4.B. compare mouse cryptdins 1-5 (SEQ ID NOS: 6 to 10,respectively) and partially purified luminal peptides.

FIG. 4.A. Lyophilized luminal lavage of small intestine from 12 mice and20 μg protein was fractionated by P-60 gel filtration andelectrophoresed on an acid-urea acrylamide gel (lane 2) along side asimilarly prepared sample of bowel tissue (lane 1). The positions ofcryptdins 1-5 (SEQ ID NOS: 6 to 10, respectively) are indicated.

FIG. 4.B. Partially purified luminal peptides (20 μg; as for FIG. 4.A.,lane 2) were electrophoresed in a second acid-urea gel (lane 3) alongwith an identical sample previously treated with performic acid (lane4). In lane 4, rapidly migrating, cyst(e)ine-containing peptides areabsent due to the increased net negative charge resulting from theconversion of cyst(e)ines to cysteic acid residues.

FIG. 5 shows the identification of mouse cryptdins 1-5 (SEQ ID NOS: 6 to10, respectively) in small intestine epithelium. Acid extracts ofintact, whole small intestine (W) or epithelial sheets (E) werelyophilized, dissolved in sample solution and resolved on a 12.5%acid-urea acrylamide gel. Cryptdins 1-5 (SEQ ID NOS: 6 to 10) areidentified numerically.

FIGS. 6.A. to 6.F. show the immunohistochemical localization of cryptdin1 (SEQ ID NO: 6) in small intestine. Full thickness sections of adultmouse jejunem were incubated with preimmune (FIGS. 6.A., 6.C. and 6.E.)or anti-cryptdin C rabbit IgG (FIGS. 6.B., 6.D. and 6.F.) and developedusing peroxidase anti-peroxidase secondary antibody magnifications:FIGS. 6.A. and 6.B., 40×; FIGS. 6.C. and 6.D., 250×; FIGS. 6.E. and6.F., 640×.

FIGS. 7.A. and 7.B. depict the antimicrobial activity of mouse cryptdin1 (SEQ ID NO: 6). Samples of purified natural mouse cryptdin 1 (FIG.7.A.) or rabbit NP-1 (FIG. 7.B.) were dissolved in 0.01% acetic acid andpipetted into wells produced in a 0.6% agarose/0.3% tryptone platecontaining 1×10⁶ log phase bacterial cells. After incubation at 37° C.for 18 hr, antimicrobial activity was evaluated by measuring thediameters of the clear zones. Closed circles denote wild type S.typhimurium; open circles denote the phoP⁻ mutant.

FIG. 8 shows the amino acid sequences for rat cryptdins 1-3 (SEQ ID NOS:15-17, respectively), human cryptdins 5 and 6 (SEQ ID NOS: 18 and 19;HD-5 and HD-6) and a consensus sequence (Def consensus). Also shown arethe amino acids sequences for rat prepro-cryptdins 1-3 (SEQ ID NOS:20-22) as deduced from cDNA or genomic DNA sequences as indicated.

FIGS. 9.A. and 9.B. show the amino acid sequences of mouse cryptdins1-17 (SEQ ID NOS: 23 to 39, respectively) as determined from the cDNAsequences encoding the specific cryptdin.

FIG. 9.A. shows the entire amino acid sequence of the mouse cryptdins.The amino acid sequences of cryptdins 1-6 (SEQ ID NOS: 23 to 28) weredetermined by sequencing the purified peptides. The amino acid sequencesof cryptdins 7-17(SEQ ID NOS: 29 to 39) were deduced from the cDNAsequences (see FIG. 10). The amino acids encoded by Exon 1, whichencodes the signal peptide and propiece, and Exon 2, which encodes themature cryptdin peptide, are indicated. A dot indicates the sequence wasnot encoded by the cDNA clone. "*" indicates a space, which preservesthe homology of the sequences.

FIG. 9.B. indicates the degree of relatedness of the mouse cryptdins.Amino acids that are identical to the amino acid shown for cryptdin 1(SEQ ID NO: 23) are indicated by a dot.

FIG. 10 shows the nucleic acid sequences for the cDNA sequences encodingmouse cryptdins 1-17 (SEQ ID NOS: 40 to 56, respectively). A consensusnucleotide sequence also is shown (SEQ ID NO: 57). A dot indicates thenucleotide is the same as shown for cryptdin 1. The amino acid sequencefor cryptdin 1 (SEQ ID NO: 23) is shown above the nucleic acid sequence.Numbers below the nucleotide sequence indicate the nucleotide positionrelative to the methionine start codon (+1). Numbers above the aminoacid sequence indicate the amino acid position. Italics indicate themature cryptdin peptide sequence. Nucleotides in lower case lettersindicate non-coding sequences. "***" indicates a stop codon. "(A)_(n) "indicates poly-A tail. "*" indicates a space and "-" indicates theparticular nucleotide could not be determined unambiguously.

FIG. 11 shows the genomic DNA sequences for mouse cryptdins 1, 2, 3, 5and 6 (SEQ ID NOS: 58 to 62, respectively) and the genomic sequence forthe apparently inactivated mouse cryptdin i gene (Crypi; SEQ ID NO: 63),in which a stop codon (TGA) is substituted for a cysteine residue.Numbering is as described in the legend to FIG. 11. The upper sequencerepresents a consensus cryptdin gene sequence (SEQ ID NO: 64). "X"indicates positions at which at least two sequences containingnucleotide changes. The TATAAA box is shown in lowercase italics; exonsare shown in capital letters; "**" indicates intron DNA; "n" representsapproximately 500 base pairs that were not sequenced. "@" indicates thestart of the cryptdin peptide coding region at codon 59. Codingsequences are indicated in bold print. Prepro-regions are coded bynucleotides 1-172; cryptdin peptides are coded by nucleotidase 173-279.The stop codon is underlined.

GenBank accession numbers for these sequences are

002994 (cryptdin 1, exon 1); 002995 (cryptdin 1, exon 2);

002996 (cryptdin 2, exon 1); 002997 (cryptdin 2, exon 2);

002998 (cryptdin 3, exon 1); 002999 (cryptdin 3, exon 2);

003000 (cryptdin 5, exon 1); 003001 (cryptdin 5, exon 2);

003002 (cryptdin 6, exon 1); 003003 (cryptdin 6, exon 2);

003004 (cryptdin i, exon 1); and 003005 (cryptdin i, exon 2).

FIGS. 12.A. to 12.C. demonstrate the effectiveness of mouse cryptdins(as indicated) in inhibiting the growth of E. coli ML35 cells in an agardiffusion assay.

FIGS. 13.A. to 13.C. demonstrate the effectiveness of mouse cryptdins(as indicated) in killing E. coli cells in suspension.

FIGS. 14.A. to 14.C. show the cDNA sequences encoding rat cryptdin 1(FIG. 14.A.), rat cryptdin 2 (FIG. 14.B.) and rat cryptdin 3 (FIG.14.C.). Nucleotide numbers are indicated.

FIGS. 15.A. to 15.C. show the genomic DNA sequences encoding ratcryptdin 1 (FIG. 15.A.), rat cryptdin 2 (FIG. 15.B.) and rat cryptdin 3(FIG. 15.C.). Nucleotide numbers are indicated.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides small peptide molecules, termed cryptdins, whichexpress a broad range of antimicrobial activity, particularly againstintestinal pathogens, and for this reason are useful antimicrobialagents. For example, cryptdins have antimicrobial activity against gramnegative and gram positive bacteria and against protozoan pathogens (seeExample III). Cryptdin peptides and nucleic acid sequences encodingcryptdins were isolated from the small intestine and are active withinthe epithelial lining of the small intestine and within the lumen of theintestine. Because it is indicative of inflammatory processes, thepresence of cryptdins can be utilized in the diagnosis of a wide rangeof inflammatory conditions.

As used herein, the term "cryptdin" or "enteric defensins" refers topeptides having generally between about 30 and 45 amino acids. Cryptdinsare characterized, in part, by a consensus sequence containing sixcysteine residues. Illustrative sequences are provided in FIG. 1, whichshows invariant residues and the disulfide bonding motif. In addition,those residues which are preferably invariant are identified (see, also,FIGS. 8 and 9).

Cryptdins are further characterized by their cationic charge and theirbroad range of antimicrobial activity. While related toleukocyte-derived defensins, cryptdins are distinguished from theseother molecules by the presence of 3 to 9 amino acids N-terminal to thefirst cysteine molecule. Cryptdins may have C-terminal extensions aswell. In addition, they exhibit antimicrobial activity against entericmicroorganisms, which can become blood-borne pathogens if the intestinalbarrier is breached. Since cryptdins are associated with the secretorygranules of Paneth cells. in the small intestine, they can be secretedfrom the cells in which they are produced (Satoh, Cell Tiss. Res.251:87-93 (1988); Satoh et al., Acta Histochem. 83:185-188 (1988)).Unlike leukocyte-derived defensins, cryptdins are not toxic to mammaliancells.

It should be appreciated that various modifications can be made to thecryptdin amino acid sequence without diminishing the antimicrobialactivity of the peptide. It is intended that peptides exhibiting suchmodifications, including amino acid additions, deletions orsubstitutions are within the meaning of the term "cryptdin" and,therefore, within the scope of the invention. For example, cryptdinanalogs, which are devoid of one or more amino acids N-terminal to thefirst cysteine residue, are within the present invention. Such cryptdinanalogs can be synthesized using well known methods (see Example VI) orcan be purified from the intestine where they may occur naturally due,for example, to partial proteolysis of a cryptdin peptide in theintestinal lumen.

Use of the phrase "substantially pure" in the present specification andclaims as a modifier of peptide, protein or nucleic acid means that thepeptide, protein or nucleic acid so designated has been separated fromits in vivo cellular environment. As a result of the separation andpurification, the substantially pure peptides, proteins and nucleicacids are useful in ways that the non-separated impure peptides,proteins and nucleic acids are not.

The cryptdin peptides of the present invention preferably containbetween about 30 and 45 amino acids (see FIGS. 1, 8 and 9). Cryptdinscan be synthesized by methods well known in the art, such as through theuse of automatic peptide synthesizers or by well-known manual methods ofpeptide synthesis (see Example VI). In addition, they can be purifiedfrom natural sources such as small intestinal epithelium of vertebrate,preferably mammalian, origin (see Example I). Such epithelium can beobtained, for example, from rats, mice or humans using means well knownto those skilled in the art.

As disclosed herein, various cryptdin peptides were isolated fromintestinal epithelium, purified by chromatographic methods andcharacterized by electrophoresis and amino acid sequencing. Cryptdinswere identified by their rapid migration on acid-urea PAGE and by theirapparent molecular weight of about 4 kDa (see Examples I and II).

Anti-cryptdin antibodies were made using methods conventional in theart. For example, polyclonal antiserum can raised in appropriateanimals, such as rabbits, mice or rats. Cryptdin peptides, eithersynthetic or obtained from natural sources, can be used to immunize theanimal. As described in Example IV, a cryptdin analog, cryptdin C, whichcorresponds to residues 4-35 of mouse cryptdin I (SEQ ID NO: 6) as shownin FIG. 1, was used to immunize rabbits using well known methods. Serumsamples were collected until the anti-cryptdin titer was appropriate.Various fractions of the antiserum, such as IgG, can be isolated bymeans well known in the art. Cryptdin immunogens also can be used toobtain monoclonal antibodies using methods well known in the art (see,for example, Harlow and Lane, Antibodies: A Laboratory Manual (ColdSpring Harbor Laboratory Press 1988), which is incorporated herein byreference).

The antimicrobial activity of a cryptdin can be measured against variouspathogens. As disclosed in Example III, various microorganisms weregrown to an appropriate concentration, mixed with an appropriate mediumsuch as an agarose-trypticase soy medium and contacted with a cryptdin.Antimicrobial activity was apparent, for example, from the clear zonesthat surrounded the cryptdins in an agar diffusion assay. The area ofthe clear zones was concentration dependent (see FIG. 12).

Anti-cryptdin antibodies can be used to determine the presence ofcryptdin in a biological sample such as a histological sample. Forexample, sections of small intestine are fixed by means well known tothose skilled in the art and incubated with anti-cryptdin antibodiessuch as an IgG fraction of antiserum. If desired, the anti-cryptdinantibody can be detectably labelled or an appropriate detectable secondantibody can be used to identify the presence of the primary antibodyattached to the cryptdin. Means of detection include the use ofradioactive protein A or enzyme substrates such as peroxidase (seeHarlow and Lane, supra, 1988).

Alternative methods of determining the presence of cryptdin in abiological sample obtained, for example, by intestinal lavage or bydisrupting cells or tissues can be useful to determine the presence ofinflammatory processes. In the presence of inflammatory processes, theconcentration of cryptdins is significantly altered from that found inthe normal cell. In particular, a deviation from the normal level ofcryptdins by one to two standard deviations is indicative of aninflammatory process. Such an inflammatory process can include, forexample, inflammatory bowel disease, pancreatitis, malignancy, infectionor ileitis.

Because of their broad range of antimicrobial activity and their abilityto function within the intestinal epithelium or lumen, cryptdins arepotent therapeutic agents for infections of the intestine. Inparticular, cryptdins are useful where the subject is immunocompromiseddue, for example, to malignancy, malnutrition, chemotherapy, radiation,immunosuppressive viruses, autoimmune disease or neonatality. Inaddition, cryptdins are useful in surgical prophylaxis, for example, byfunctioning to help sterilize the small bowel. Thus, cryptdins can beuseful as medicaments for treating a subject having a pathologycharacterized, in part, by an inflammatory process.

A cryptdin, either purified from natural sources or synthetic, can beadministered to a subject in need of such therapy by various means,including orally, preferably in a slow-release type formulation, whichwill avoid release within the stomach. Alternatively, cryptdins can beadministered through nasogastric intubation, transabdominal catheter,intravenously or aerosol administration. Individual species of cryptdincan be administered alone or in combination. Cryptdins administered incombination can be administered simultaneously or sequentially and canbe repeated as necessary.

Prior to the characterization of a mouse intestinal defensin cDNA,expression of defensins was thought to be limited to professionalphagocytes, i.e., neutrophils and macrophages. The presence of highlevels of cryptdin mRNA in Paneth cells led to the hypothesis thatdefensins synthesized in intestinal epithelium may contribute toantimicrobial barrier function in the small bowel (Ouellette et al., J.Cell Biol. 108:1687-1695 (1989a), which is incorporated herein byreference). Isolation and characterization of six mouse cryptdinpeptides, two rat cryptdin peptides and 2 human cryptdin peptides, andthe demonstration of antimicrobial activity of various cryptdin peptidesindicates that the cryptdins have an antimicrobial role in the smallintestine. The immunohistochemical localization of cryptdin(s) to Panethcells is consistent with previous in situ hybridization analysis andsuggests that defensins produced by these cells may contribute torestricting the colonization and invasion of the small bowel bybacteria.

Initial efforts to purify intestinal defensins focused on the isolationof mouse cryptdin 1 (SEQ ID NO: 6), the peptide predicted from thecryptdin cDNA sequence. Since the deduced structure of the peptide ishighly cationic, intestinal peptides were solubilized by homogenizingintact mouse jejunum and ileum in 30% formic acid. Acid-urea PAGE of thecrude extract revealed several bands with R_(f) values similar to thoseof rabbit defensin NP-1 and cryptdin C, a folded synthetic defensincongener corresponding to residues 4 to 35 in cryptdin 1 (SEQ ID NO: 6).Peptides corresponding to these bands were purified approximately200-fold by sequential gel filtration chromatography on Bio-Gel P-60(FIGS. 2.A. and 2.B.). Electrophoresis of P-60 column fractions onacid-urea gels showed that five fractions eluting between two prominentpeaks (FIGS. 2.A. and 2.B., brackets) contained putative cryptdinpeptides (FIG. 3, lane a). Peptides in these P-60 fractions migratedwith an apparent molecular mass of approximately 4 kDa on SDS-PAGE (notshown), consistent with the molecular weight of defensins. Furthermore,treatment of P-60 fraction samples with performic acid reduced theelectrophoretic mobility of the five putative mouse cryptdins inacid-urea gels, behavior that is characteristic of defensins andpolypeptides that contain multiple cysteine residues.

Defensins in pooled P-60 fractions were purified further usingsequential rounds of RP-HPLC utilizing different ion-pair agents.Initial HPLC fractionation utilized water-acetonitrile gradientscontaining 0.13% heptafluorobutyric acid (HFBA) as the ion-pairingagent, whereby each of the five peptides contained in the pooled P-60fractions was resolved to near purity in a single run (FIG. 2.C.).Complete purification of five peptides, mouse cryptdins 1-5 (SEQ ID NOS:6 to 10, respectively), was achieved by subsequent RP-HPLC using 0.1%trifluoroacetic acid (TFA) (FIG. 3, lanes B-F). Assuming extraction ofindividual peptides is equally efficient, both acid-urea gelelectrophoresis and RP-HPLC of the P-60 fractions containing putativecryptdins showed that the relative abundance of the peptides is cryptdin1 > cryptdin 2 > cryptdin 5 > cryptdin 3 > cryptdin 4. The relativeamounts of cryptdins 1-5 (SEQ ID NO: 6 to 10, respectively) have beenqualitatively reproducible in every preparation of acid-extractedprotein from mouse small intestine.

Using a modification of the method described above, mouse cryptdin 6,rat cryptdin 2, and human cryptdins 5 and 6 also were isolated (seeExamples I and II; see, also, FIGS. 8 and 9). In addition, longer formsof mouse cryptdins 4 and 5 (compare SEQ ID NOS: 9 and 12; 10 and 13) andrat cryptdin 1 (compare SEQ ID NOS: 11 and 15) were obtained. Thisresult suggests that the initial method of purifying cryptdin peptidesresulted in partial degradation of the C-termini of some peptides.Significantly, both forms of the purified cryptdin peptides haveantimicrobial activity.

Biochemical characterization of the isolated cryptdins demonstrated thatthese peptides are defensins. Amino acid analysis of each peptide showedtheir compositions (cationic peptides of about 30 to 45 amino acidresidues, including 6 half-cysteines) are compatible with defensin-likemolecules. The complete sequences of mouse cryptdins 1-6 (SEQ ID NOS: 23to 28), rat cryptdins 1 and 2 (SEQ ID NOS: 11, 15 and 16) and humancryptdins 5 and 6 (SEQ ID NOS: 18 and 19) were determined by automatedEdman degradation and, in some cases, by amino acid analysis of carboxylterminal chymotryptic peptides (see FIGS. 1, 8 and 9). The primarystructures of the cryptdins contain the distinctive structural featuresof human, rabbit, rat and guinea pig neutrophil defensins (Lehrer etal., Cell 64:229-230 (1991a), which is incorporated herein byreference), i.e., the six invariant cysteine residues and the glycineand glutamic acid in positions that are highly conserved in myeloiddefensins.

The cryptdin peptides disclosed herein contain features that are uniqueand distinct from defensins of myeloid origin. For example, mousecryptdins 1, 2, 3 and 6 (SEQ ID NOS: 23 to 25 and 28, respectively) arealmost identical, differing only at two or three positions (see FIG.9.A.). Analysis of codons from which these amino acid differences couldarise shows that the conversion, for example, of Ser¹⁰ to Lys¹⁰ incryptdin 1 (SEQ ID NO: 23) and cryptdin 3 (SEQ ID NO: 25), respectively,requires two nucleotide substitutions. On the other hand, singlenucleotide changes in the codon encoding Thr¹⁰ in cryptdin 2 (SEQ ID NO:24) could give rise to cryptdins 1, 3 and 6, suggesting that thecryptdin 2 gene may be an intermediate or progenitor of the cryptdin 1,3 and 6 genes. Similarly, a single nucleotide change in the codon forThr¹⁰ of cryptdin 2 can account for the deduced amino acid at position10 in cryptdins 7-17 (see FIG. 10, nucleotides 203-205; SEQ ID NOS: 46to 56).

By homology with the structures of known myeloid defensins, the cryptdin1 N-terminus was predicted to begin at Leu⁴ or Val⁵, which is 1-2residues prior to the first conserved cysteine. However, compared tomyeloid defensins, cryptdins have variably extended N-termini that acontain from three (mouse cryptdin 4, SEQ ID NO: 26; rat cryptdin 1, SEQID NO: 11) to nine (human cryptdin 5, SEQ ID NO: 18) amino acidspreceding the first cysteine. In mouse cryptdins 1-3 and 6-17 (SEQ IDNOS: 23 to 25 and 28 to 39, respectively), the N-peptidyl extensionsconsist of two charged internal residues flanked by amino acids withhydrophobic sidechains. Since natural variation in defensin aminotermini correlates with relative antimicrobial potency in vitro (Ganz etal., J. Clin. Invest. 76:1427-1435 (1985), which is incorporated hereinby reference), the extended N-termini of enteric defensins may haveevolved for a unique role in the bowel.

Mouse cryptdin 4 (SEQ ID NO: 9), the most cathodal and, apparently,least abundant mouse enteric defensin, was the first defensin found tocontain a chain length variation between the fourth and fifth cysteineresidues. Unlike the majority of previously known defensins, in whichnine amino acids separate the fourth and fifth cysteines (Lehrer et al.,supra, 1991a), mouse cryptdin 4 (SEQ ID NO: 9) contains only sixresidues between the same two amino acids (FIG. 1). In addition, ratcryptdins 1-3 (SEQ ID NOS: 11 and 15-17) contain ten amino acid residuesbetween the fourth and fifth cysteines. These findings indicate thedefensin fold involving this stretch of the peptide chain canaccommodate significant variability in the size of the loop, as comparedto the invariant loop size defined by crystal and NMR structures,respectively, of human and rabbit neutrophil defensins. Also, ratcryptdins 1-3 (SEQ ID NOS: 11 and 15-17) are the only cryptdinscontaining three, instead of four, amino acid residues between thesecond and third cysteine residues.

Since cryptdin mRNA levels increase during postnatal development ofmouse small bowel (Ouellette et al., supra, 1989a), it was investigatedwhether accumulation of enteric defensins was regulated similarly.Analysis of intestinal acid extracts from male and female mice showedthat mouse cryptdins 1-3 and 5 (SEQ ID NOS: 6 to 8 and 10, respectively)are present in adult mice, regardless of gender. On the other hand,extracts from 9 day-old mice lack the peptides, consistent withpostnatal accumulation of cryptdin MRNA.

Mouse cryptdins 1-5 (SEQ ID NOS: 6 to 10) were purified from intestinalepithelial cells. In the presence of EDTA, the intestinal epithelium nolonger adheres to the underlying basement membrane and floats free ofthe lamina propria upon gentle agitation (Bjerknes and Cheng, Am. J.Anat. 160:51-63 (1981), which is incorporated herein by reference).Preparations of epithelial sheets isolated in this manner wereconcentrated by low speed centrifugation and extracted with 30% formicacid. Peptides extracted from isolated epithelial sheets comigrate withcryptdins 1-5 (SEQ ID NOS: 6 to 10) when analyzed by acid-urea PAGE(FIG. 5), demonstrating their epithelial origin.

Immunoperoxidase staining of full-thickness sections of small intestinewith an anti-cryptdin antibody demonstrate cryptdin antigen in Panethcells, consistent with localization of cryptdin mRNA by in situhybridization (Ouellette et al., supra, (1989a)). Incubation of sectionsof adult mouse jejunum and ileum with a polyclonal anti-cryptdin IgGproduced by rabbits immunized with the synthetic congener cryptdin Clocalized the immunoperoxidase reaction to granulated cells,morphologically defined as Paneth cells, at the base of every crypt(FIG. 6). The staining pattern accentuates the granular appearance ofthe cytoplasm in these cells and the immunoreactivity appearsparticularly strong over Paneth cell granules. The antibody is specificfor mouse cryptdin(s), since it is negative both for rat and humanPaneth cells (data not shown). Leukocytes in the lamina propria of thevilli also were negative, suggesting that related enteric defensins arenot expressed by phagocytes or lymphocytes. Because of the extensivesimilarity of mouse cryptdins 1-3 (FIG. 1; SEQ ID NOS: 6 to 8), thepolyclonal antibody produced against cryptdin C probably recognizes thethree peptides. Conversely, because mouse cryptdin 4 (SEQ ID NO: 9) andcryptdin 5 (SEQ ID NO: 10) differ markedly from the other mousecryptdins, the anti-cryptdin C antibody is unlikely to react withcryptdin 4 (SEQ ID NO: 9) and cryptdin 5 (SEQ ID NO: 10), leaving theirorigin in Paneth cells somewhat unresolved.

Immunohistochemical data suggest cryptdins are secreted into theintestinal lumen. Material in the small intestinal lumen is stronglypositive for the antibody but negative for pre-immune sera or IgG (FIGS.6.A. and 6.B.). Although the agonist for Paneth cell defensin secretionis unknown, lysozyme, another protein constituent of Paneth cellgranules, is secreted into the lumen under cholinergic regulation.Consistent with immunochemical detection of anti-cryptdin C positivematerial in the intestinal lumen, acid-urea PAGE of saline washes ofadult jejunum and ileum contain peptides with mobilities very similar tobut distinct from the mobility of cryptdins (FIG. 4). Nevertheless, thepeptides are not identical to cryptdins 1-5 (SEQ ID NOS: 6 to 10,respectively) by either migration in acid-urea PAGE or by HPLC analysis,suggesting they may correspond to cryptdins that have been processedfurther. Conceivably, luminal cryptdin or cryptdin-like material couldderive from exfoliated Paneth cells in the lumen, but the low rate ofPaneth cell turnover suggests this is unlikely. The release of cryptdinsor processed variants into the small bowel by Paneth cells contrastswith the apparent lack of defensin secretion by leukocytes, and it isinferred that a secretory pathway may exist for the constitutivedelivery of defensins into the intestinal lumen by Paneth cells.

The antibacterial activity of purified mouse cryptdins 1-5 (SEQ ID NOS:6-10) was tested against wild type and phoP mutant S. typhimurium usinga modified plate diffusion assay (Lehrer et al., J. Immunol. Methods137:167-173 (1991b), which is incorporated herein by reference). phoP isa two-component regulatory locus that is essential to S. typhimuriumvirulence and survival within macrophages (Fields et al., Science243:1059-1062 (1989); Miller et al., Proc. Natl. Acad. Sci.. USA86:5054-5058 (1989), each of which is incorporated herein by reference).Mutants in the phoP locus are particularly sensitive to rabbit defensinsNP-1 and NP-2 when compared to wild type parent strains (Fields et al.,supra, 1989; Miller et al., Infect. Immun. 58:3706-3710, (1990), whichis incorporated herein by reference).

Under assay conditions using a phosphate buffer as described in ExampleIII, the antimicrobial activity of rabbit defensin NP-1 against wildtype and the phoP mutant organisms was quite similar (FIG. 7.B.). On theother hand, at concentrations of mouse cryptdin 1 (SEQ ID NO: 6) thatare effective against the attenuated mutant, wild type S. typhimurium iscompletely resistant to the effects of the peptide (FIG. 7.A.).

The differential activity of cryptdin 1 (SEQ ID NO: 6) against avirulentS. typhimurium suggests that resistance to mucosal defensins may beimportant for the evolution of virulence in enteric pathogens. However,in experiments using HEPES or PIPES as buffers as described in ExampleIII, concentrations of 100 μg/ml or 300 μg/ml cryptdin 1 were aseffective as NP-1 in inhibiting the growth of wild type S. typhimurium.Furthermore, at these concentrations, cryptdins 4 and 5 were moreeffective than NP-1 in preventing the growth of mutant and wild type S.typhimurium (not shown).

The present invention also provides substantially purified nucleic acidsequences encoding cryptdins. For example, the cDNA sequences for mousecryptdins 1-17 (SEQ ID NOS: 40-56) are shown in FIG. 10 and the cDNAsequences for rat cryptdins 1-3 (SEQ ID NOS: 65-67) are shown in FIGS.14.A. to 14.C. In addition, the genomic DNA sequences for mousecryptdins 1, 2, 3, 5 and 6 (SEQ ID NOS: 58-62) and for an apparentlyinactivated cryptdin gene, cryptdin i (SEQ ID NO: 63) are shown in FIG.11 and the genomic DNA sequences for rat cryptdins 1-3 (SEQ ID NOS:68-70) are shown in FIGS. 15.A. to 15.C.

The skilled artisan would recognize that various nucleotidesubstitutions could be made in the nucleic acid sequences shown in FIGS.10, 11 14 and 15 without altering the amino acid sequence of the encodedcryptdin peptide due to degeneracy of the genetic code. Such nucleotidesequences, which are equivalent to the sequences shown in FIGS. 10, 11,14 and 15 are encompassed within the claimed invention.

The invention also provides nucleotide sequences that consist of aportion of a nucleic acid sequence as shown in FIGS. 10, 11, 14 and 15.Such a nucleotide sequence can be useful, for example, as a probe, whichcan hybridize under relatively stringent conditions to a nucleic acidmolecule encoding a cryptdin peptide. For hybridization, such anucleotide sequence should be at least about 10 nucleotides in length.One skilled in the art would know that appropriate conditions forhybridization can be determined empirically or can be calculated based,for example, on the G:C content of the nucleotide sequence, the lengthof the sequence and the number of mismatches, if any, between the probeand the target sequence (see, for example, Sambrook et al., MolecularCloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989),which is incorporated herein by reference).

A nucleotide sequence as described above can be detectably labelled byattaching, for example, a radioactive label or biotin, or can beunlabelled. A labelled or unlabelled sequence also can be used as aprimer for the polymerase chain reaction (PCR; see, for example, Erlich,PCR Technology: Principles and applications for DNA amplification(Stockton Press 1989), which is incorporated herein by reference). Sucha sequence can be useful, for example, to identify a nucleic acidsequence encoding a cryptdin in a cell.

A nucleic acid molecule as shown in FIGS. 10, 11, 14 and 15 or anucleotide sequence derived therefrom also can be useful, for example,for preparing a cryptdin peptide or a portion of a cryptdin peptideusing well known methods of recombinant DNA technology. For example, thenucleic acid sequence can be cloned into an expression vector such as abaculovirus vector or a viral vector, which can infect a mammalian celland express an encoded cryptdin peptide in the cell. Expression fromsuch a vector can be useful for producing large amounts of a cryptdin,which can be used to treat a subject having an inflammatory pathology asdescribed herein, or for producing a cryptdin directly in a subject.Thus, the invention provides vectors containing a nucleic acid moleculeas shown in FIGS. 10, 11, 14 and 15 as well as specific host cells, inwhich the vector can propagate or can express a cryptdin.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE I Purification of Enteric Defensins

Outbred Swiss mice, (Crl:CD-1) (ICR)BR, 45 day old males (30-35 g) ortimed-pregnant dams, were obtained from Charles River BreedingLaboratories, Inc. (North Wilmington Mass.). In studies of newborn mice,litters were culled to 8 pups within 12 hr of delivery. Mice were housedunder 12 hr cycles of light and darkness and had free access to food andwater.

Cryptdins were isolated by a modification of the method described bySelsted et al., J. Cell. Biol. 118:929-936 (1992); Ouellette et al.,Infect. Immun. 62:5040-5057 (1994), each of which is incorporated hereinby reference. Jejunal and ileal intestinal segments were excised from 60mice immediately after carbon dioxide euthanasia. The tissue was washedand the lumen was flushed with ice cold water prior to homogenization in500 ml ice cold 30% acetic acid. The homogenate was clarified bycentrifugation, lyophilized, dissolved in 200 ml 30% acetic acid,clarified by filtration through Whatman 541 filter paper and applied toa 10×60 cm Bio-Gel P-60 column equilibrated with 30% acetic acid. Theelution rate was 100 ml/hr. Fractions containing cryptdins wereidentified by electrophoresis in acid-urea polyacrylamide gels (Selstedand Harwig, Infect. Immun. 55:2281-2285 (1987), which is incorporatedherein by reference).

Cryptdin-containing fractions were pooled and lyophilized, thenpurification was completed by RP-HPLC. Initial separation of mousecryptdins 2-5 was achieved by HPLC on a 1×25 cm Vydac C-18 column usinga gradient of water and acetonitrile containing 0.13% HFBA. Solventswere delivered at 3 ml/min to generate the following acetonitrilegradient: 0-28% (10 min); 28-34% (20 min); and 34-40% (60 min).Cryptdins 1 and 6, which coeluted under these conditions, were resolvedby C-18 RP-HPLC using 0.1% TFA as the ion pair and a 16-21% acetonitrilegradient delivered in 35 min at 3 ml/min. To eliminate traces ofresidual HFBA, preparations of cryptdins 2-5 were subjected to anaddition RP-HPLC step using 0.1% TFA. All peptides were lyophilized andquantitated by amino acid analysis prior to antimicrobial testing.Essentially identical methods were used to purify rat and human cryptdinpeptides, except that rat cryptdins were isolated from the smallintestine of adult Sprague-Dawley rats and human cryptdins were isolatedfrom a surgically resected normal adult human male small intestine.

EXAMPLE II Peptide Characterization

Amino acid analyses were performed on 6 N HCl hydrolysates (150° C., 2hr) of unmodified or performic acid-oxidized peptides. Hydrolysates werederivatized with phenylisothiocyanate and the resultingphenylthiocarbamyl amino acids were quantitated as described previously(Selsted and Harwig, supra, 1987; Selsted et al., supra, 1992; Ouelletteet al., FEBS Lett. 304:146-148 (1992), which is incorporated herein byreference) Peptide samples were reduced with dithiothreitol (DTT) andpyridylethylated with 4-vinyl pyridine for sequencing (Henschen, InAdvanced Methods in Protein Microseguence Analysis (Wittmann-Liebold etal., pages 244-255 (1986), which is incorporated herein by reference).Sequence determinations were performed by automated Edman degradation onan ABI model 477 system (Applied Biosystems, Inc.; Foster City Calif.)with on-line PTH amino acid analysis. In some cases, the C-terminus of acryptdin peptide was confirmed by amino acid analysis of chymotrypticpeptides. Cryptdins 4 and 5 also were analyzed by positive-ion fast atombombardment mass spectrometry on a VG 7070E-HF instrument (Ouellette etal., supra, 1994).

EXAMPLE III Antimicrobial Assays

Antibacterial activity was measured in an agar radial diffusion assay(Lehrer et al., supra, 1991b) using wild type S. typhimurium (ATCC10428) or an isogenic phoP mutant of S. typhimurium (strain CS015phoP102::Tn10d-Cam, Miller et al., supra, 1989). Cells were grown to logphase in trypticase soy broth at 37° C., harvested by centrifugation andresuspended to 1×10⁷ colony forming units (CFU) per ml in 10 mM sodiumphosphate buffer (pH 7.4).

A 100 μl aliquot of each organism was mixed with 10 ml 1% agarose in0.03% (w/v) trypticase soy medium, 10 mM sodium phosphate (pH 7.4) at42° C. Five μl samples of peptide solution were pipetted into 3 mmdiameter wells formed in the agarose with a sterile punch. After 3 hr at37° C., the inoculated agarose plate was overlayed with 1% agarosecontaining 6% trypticase soy medium. After 12-16 hr, antimicrobialactivity was apparent as clear zones surrounding wells loaded withantibacterial samples; the sizes of the clear zones wereconcentration-dependent.

Cryptdin antimicrobial activity in vitro was substantially enhanced inpiperazine-N,N'-bis (2-ethane sulfonic acid) (PIPES) or inN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) as comparedto the activity in sodium phosphate. Purified cryptdin peptides weredissolved at 3 to 300 μg/ml in 0.01% acetic acid and activity wasexamined against E. coli ML35 (ATCC). In the radial diffusion assay, 5μl peptide solution was transferred into wells formed in plates of 1%agarose buffered with 10 mM PIPES (pH 7.4) and containing 1×10⁶log-phase bacteria grown in trypticase soy broth. After 3 hr at 37° C.,the plates were overlayed with 0.8% agarose containing 2× trypticase soybroth and incubated overnight. The antibacterial activities of cryptdinpeptides was compared with the activity of rabbit neutrophil defensinNP-1, which was purified from peritoneal exudates as described bySelsted et al. (J. Biol. Chem. 260:4579-4584 (1985), which isincorporated herein by reference). Antibacterial activity was determinedby measuring the diameter of clearing around each well and plotted as afunction of peptide concentration.

As shown in FIG. 12, each cryptdin peptide produced a dose-dependentzone of clearing, which indicates that E. coli growth was inhibited. Thepotencies of the cryptdins varied, with cryptdins 1, 3 and 6 showingsimilar activity, which was about 3-5× greater than the activity ofcryptdin 2. Cryptdin 5 was approximately 5× more active than rabbit NP-1at concentration above 100 μg/ml (FIG. 12.C.) and cryptdin 4 was atleast 50× more active than NP-1 when compared at 100 μg/ml and 300 μg/ml(FIG. 12.B.). These higher concentrations of cryptdins 4 and 5 also weremore effective than the same concentration of NP-1 at inhibiting thegrowth of S. aureus and of wild type and mutant strains of S.typhimurium (not shown). These results demonstrate that various cryptdinpeptides can inhibit bacterial growth.

In order to determine whether the effect of the cryptdin peptidesagainst E. coli is bacteriostatic or bacteriocidal, bacterial killingwas quantitated as a function of time. Bactericidal assays wereperformed by incubating 1-2×10⁶ log-phase bacteria in 10 mM PIPEScontaining 10 μg peptide/ml. After incubation for 15 or 30 min at 37°C., aliquots were removed, serially diluted and plated on trypticase soyagar. Bactericidal activity was quantitated by counting colonies afterovernight incubation at 37° C.

As shown in FIG. 13, cryptdins 1 and 3-6 rapidly killed the E. colicells. In each of these cases, survival was reduced to less than 1%after only 15 min incubation. Cryptdin 2 was the only peptide testedthat was not bactericidal under the assay condition. Cryptdins 2 and 3differ only at amino acid position 10 (threonine and lysine,respectively).

The bactericidal activity of rat cryptdin 1 also was examined. E. coliML35 cells, S. aureus 502A cells or mutant or wild type S. typhimuriumcells were incubated with various concentrations of rat cryptdin 1 orrabbit NP-1. Ten μg/ml rat cryptdin 1 killed about 90% of the S. aureuscells and greater than 99% of the E. coli and mutant S. typhimuriumcells, but was relatively ineffective in killing wild type S.typhimurium (not shown). Rat cryptdin 1 was more effective than NP-1 inkilling the E. coli and mutant S. typhimurium cells, whereas NP-1 wasmore effective in killing S. aureus.

The effect of mouse cryptdins 1-3 and 6 at inhibiting the growth of theprotozoan, Giardia lamblia, which is the most common cause of protozoandisease in the human small intestine, also was examined. Briefly,trophozoites of the C6 clone of Giardia lamblia WB (ATCC 30957) weregrown to late log phase in TYI-S-33 medium containing bovine bile.Free-swimming trophozoites were discarded and tubes with attachedtrophozoites were refilled with warm Dulbecco's PBS. Trophozoites weredetached by chilling 10 min on ice, then harvested by centrifugation,resuspended at 2×10⁷ /ml in 25 mM HEPES (pH 7.5) containing 9%(isotonic) sucrose and incubated for 2 hr at 37° C. with variousconcentrations of mouse cryptdins 1-3 or 6. Following incubation,trophozoite viability was determined by trypan blue exclusion.

The cryptdin peptides killed Giardia trophozoites in a dose-dependentmanner (not shown). In particular, 20 μg/ml of cryptdin 2 or cryptdin 3reduced Giardia growth by greater than 2 orders of magnitude (notshown). These results indicate that cryptdins are active against avariety of microorganisms.

EXAMPLE IV Anti-cryptdin Antibody

A polyclonal rabbit antibody was prepared to a synthetic analogue ofcryptdin 1. The peptide, termed cryptdin C, corresponding to residues4-35 in cryptdin 1 (SEQ ID NO: 6; FIG. 1) was synthesized by solid phasechemistry using N.sup.α -butoxycarbonyl protection (Kent, Ann. Rev.Biochem. 57:957-989 (1988), which is incorporated herein by reference).Following cleavage/deprotection of synthetic cryptdin C withTFA-trifluoromethanesulfonic acid, the peptide was precipitated in ethylether and dried in vacuo. A 100 mg sample was dissolved in 10 ml 6.0 Mguanidine-HCl, 0.2 M Tris-HCl, pH 8.2, containing 20 mg DTT. The samplewas purged with nitrogen, heated to 50° C. for 4 hr and diluted 100-foldwith deionized water, then was dialyzed exhaustively, first against 0.1M sodium phosphate (pH 8.2), 20 mM guanidine-HCl, 100 mM NaCl, thenagainst 5% acetic acid. The sample was lyophilized, dissolved in 10 ml5% acetic acid and subjected to RP-HPLC on a 1×25 cm Vydac C-18 column.The earliest eluting peak, representing about 0.5% of the crude peptide,was determined by amino acid analysis to have the desired composition.

A sample (1.5 mg) of cryptdin C was supplied, without conjugation tocarrier, to Berkeley Antibody Company (Berkeley, Calif.) forimmunization of 2 New Zealand White rabbits. Serum samples werecollected for 12 weeks, until the anti-cryptdin C titer, determined byELISA, reached about 1:10,000 for each rabbit. IgG was isolated fromantiserum using DEAE Econo-Pac chromatography (Bio-Rad; Richmond Calif.)as described by the manufacturer.

EXAMPLE V Immunohistochemistry

Paraffin sections of formalin-fixed mouse mid-small bowel weredeparaffinized, treated with 1.1% hydrogen peroxide for 40 min, thenwashed extensively with water followed by PBS. Slides were treated for20 min at 37° C. with 500 μg/ml trypsin in PBS, washed twice with PBS,and blocked by incubation for 20 min with 5% porcine serum. Slides wereincubated for 20 min in rabbit anti-cryptdin IgG (1:10 dilution relativeto serum IgG concentration), then washed with blocking serum. Porcineanti-rabbit IgG was used as linking reagent between the primary antibodyand rabbit antiperoxidase-peroxidase conjugate (Dako; CarpinteriaCalif.). Diaminobenzidine was used as peroxidase substrate and parallelincubations were performed using equivalent dilutions of rabbitpreimmune IgG as the primary antibody.

EXAMPLE VI Preparation of Synthetic Cryptdin 1

This example provides a method for synthesizing, purifying andcharacterizing synthetic cryptdin 1.

A. Synthesis

Synthesis was initiated at the 0.13 mmole scale using Wang resin coupledto flourenylmethoxycarbonyl (Fmoc)-arginine using an acid labile linker.Synthesis was carried out in dimethylformamide (DMF) using (relative toresin substitution) a 3-fold excess of Fmoc-amino acids activated insitu with a 3-fold excess of BOP (benzotriazol-1-yl-oxy-tris(dimethylamino) phosphonium hexafluorophosphate) and HOBt(hydroxybenzotriazole) and a 6-fold molar excess of N-methylmorpholine(Nmm). Fmoc removal during synthetic cycles was achieved using cycles of50% and 25% piperidine in DMF. The side-chain protection scheme utilizedthe following Fmoc-amino acids: OtBut-aspartic acid, Pmc-arginine,tBut-tyrosine, tBut-serine, Trt-cysteine, tBoc-lysine, OtBut-glutamicacid, Trt-asparagine, tBut-threonine and Trt-histidine.

The peptide chain was assembled in a Synostat batch synthesizer usingsingle couplings at all additions except at leucine and valine whichwere double coupled. The cycle sequence is as follows:

1. Wash with DMF 4× for 2 min;

2. Deblock: 50% piperidine 1× for 5 min;

3. Deblock: 25% piperidine 1× for 15 min;

4. Wash with DMF 4× for 2 min;

5. Dissolve amino acids + BOP + HOBt in DMF and transfer to reactionvessel;

6. Add Nmm to RV and mix for 60 min; and

7. Wash with DMF 1× for 2 min.

After coupling of the amino terminal residue, the terminal Fmoc groupwas removed using the following cycle:

1. Wash with DMF 4× for 2 min;

2. Deblock: 50% piperidine 1× for 5 min;

3. Deblock: 25% piperidine 1× for 15 min;

4. Wash with DMF 4× for 2 min;

5. Wash with dichloromethane 1× for 5 min;

6. Wash with isopropanol 4× for 5 min;

7. Dry under stream of N₂ 1× for 10-20 min; and

8. Dry under vacuum 1× for 12 hr.

The peptide-resin was weighed to determine mass increase. To cleave anddeprotect the peptide-resin, it was first reswelled in dichloromethane,then cleaved and deprotected by addition of reagent R (90%trifluoroacetic acid, 5% thioanisole, 3% ethanedithiol, 2% anisole) at aratio of 10 ml/g peptide-resin. Cleavage/deprotection was carried outunder nitrogen for 18 hr at RT.

B. Purification

The cleavage mixture was separated from resin by filtration through ascintered glass funnel, washed with 1-2 ml fresh reagent R and diluted5-fold with 50% acetic acid. Glacial acetic acid was added to a finalacetic acid concentration of 50%. The resulting solution was extracted3× with 0.33 vol methylene chloride and the aqueous phase waslyophilized to dryness, then dissolved in 50% acetic acid andrelyophilized. The extraction and lyophilization steps were repeated 3-4times, then the dry peptide was dissolved in 30% acetic acid at aconcentration of 20 mg/ml and passed over an 800 ml Sephadex G-10 columnequilibrated in 30% acetic acid. Peptide-containing fractions werepooled, lyophilized, dissolved in 5% acetic acid, then diluted ten-foldwith water to a final protein concentration of about 1 mg/ml. Thesolution was adjusted to pH 8.0 with ammonium hydroxide and mixedrapidly with a magnetic stirrer at RT in a beaker open to room air. ThepH was adjusted periodically to pH 8.0 over a period of 4 days. Thesolution was then acidified with acetic acid to pH 3.5 and lyophilized.

C-18 RP-HPLC using 0.1% TFA-water/acetonitrile gradients was used topurify the folded peptide. Fractions were analyzed on acid-urea gels andcompared to natural cryptdin 1. The yield from an initial crude peptidepreparation of 500 mg was approximately 30 mg.

C. Characterization

Synthetic cryptdin 1 was compared to natural peptide on analyticalRP-HPLC, SDS-PAGE and under three different conditions on acid-ureaPAGE. For analysis on acid-urea PAGE, peptide was electrophoresed eitherwithout modification, after reduction with DTT or after performic acidoxidation. Under all conditions described, native and synthetic cryptdin1 behaved identically. The amino acid compositions of natural andsynthetic cryptdin 1 were indistinguishable.

EXAMPLE VII Cloning of Nucleic Acid Molecules Encoding Cryptdins

Individual crypts were isolated using a modification of the EDTA elutionmethod of Bjerknes and Cheng, supra, 1981, as described by Cano-Gauci etal., Expt. Cell Res. 208:344-349 (1993), which is incorporated herein byreference. Briefly, the central 10 cm of small intestine from an adultC3H/HeJ mouse was everted on a Buchler gradient-making apparatus, thenintact crypts were dislodged by vibration in ice cold 30 mM EDTA incalcium-free, magnesium-free PBS. Isolated crypts were disrupted in asonicating water bath prior to cDNA synthesis.

The crypt library was constructed by mRNA-directed PCR amplification(Cano-Gauci et al., supra, 1992). Phage were screened at a density ofapproximately 300 PFU/dish using the partial cDNA clone, asb4/134, as aprobe (Ouellette et al., supra, 1989a). Positive phage were collectedand denatured plasmid cDNA was sequenced by the dideoxynucleotidetermination method using Sequenase™ (U.S. Biochemical Corp.; ClevelandOhio). Sequencing primers included T3 and T7 promoter primers andDefcr_(p) ₁₃₀, which is a 16-mer that corresponds to nucleotides 90-105in cryptdin 1 mRNA (Huttner et al., Genomics 19: 448-453 (1994), whichis incorporated herein by reference). Reaction mixtures were separatedby electrophoresis in gels consisting of 5% Long Ranger™ (AT Biochem,Inc.; Malvern Pa.) and DNA sequence data were analyzed (Ouellette etal., supra, 1994). Computations for similarity searches of DNA sequencesin nonredundant nucleic acid and protein sequence databases wereperformed at the National Center for Biotechnology Information with theBLAST network service (Ouellette et al., supra, 1994).

A cDNA library also was prepared by amplification of cryptdin mRNA(Huttner et al., supra, 1994). Total RNA was isolated from the smallintestine of a male 129/SVJ mouse using RNazol™ (Biotecx Lab; HoustonTex.). First strand cDNA synthesis was performed using the cDNA CycleKit (Invitrogen; San Diego Calif.). Amplification of 5' ends wasperformed using the 5' RACE method (Frohman et al., Proc, Natl. Acad.Sci., USA 85:8998-9002 (1988), which is incorporated herein byreference) with a reverse primer that was specific for a conservedregion of the cryptdin 3'-untranslated sequence (UTS).

Blot hybridization of the PCR products using an oligonucleotide probespecific for the cryptdin prepro-coding region detected a single band.DNA from the band was isolated using the Geneclean II™ kit (Bio101; LaJolla Calif.), subcloned into the Bluescript II plasmid using thepCR-Script SK(+) cloning kit (Stratagene) and transfected into competentXL-1 Blue cells (Stratagene). Colonies containing cryptdin-relatedsequences were identified by hybridization to a labelled asb4/134 probe.DNA sequence analysis of the positive clones was performed as describedabove, except that internal primers were utilized as required.

Using these methods, cDNA sequences encoding 17 distinct mouse cryptdinpeptides were identified (FIG. 10; SEQ ID NOS: 40-56). The various mousecryptdin cDNA sequences share 93-100% nucleotide sequence identity withcryptdin 1, except cryptdin 5 and cryptdin 4 share 73% and 69% sequenceidentity, respectively, with cryptdin 1.

The amino acid sequences were deduced from the cDNA sequences for the 17mouse cryptdins (see FIG. 9.A.; SEQ ID NOS: 23 to 39). As shown in FIG.9.A., the cDNA sequences encode prepro-cryptdin peptides consisting of asignal peptide, a propiece and the cryptdin peptide. Theprepro-cryptdins, including the mature cryptdin peptide, sharesignificant amino acid sequence identity with cryptdin 1, althoughcryptdins 4 and 5 are less homologous (FIG. 9.B.). Amino acidvariability was most striking at position 10 of the mature cryptdinpeptide, where either serine, threonine, alanine, isoleucine or lysinecan be found. Interestingly, a single nucleotide change in the sequenceof cryptdin 2 can account for each of these amino acids. In addition,position 15 can contain arginine or lysine. The amino acid variabilityamong cryptdin peptides can be involved in conferring differentantimicrobial properties to the cryptdins.

Mouse cryptdin genomic clones also were obtained and sequenced (Huttneret al., supra, 1994). Asb4/134 was used as a probe to screen acustom-made 129/SVJ mouse genomic library constructed in lambda DASH II(Stratagene Cloning Systems, Inc.; La Jolla Calif.). Approximately 1×10⁶phage were screened in duplicate and 25 positive phage were identified.Ten clones were purified and phage DNA was isolated using Qiagen 100columns (Qiagen, Inc.; Chatsworth Calif.). Southern blots of EcoRI-digested DNA from individual phage were hybridized to asb4/134 andhybridizing fragments were subcloned into Bluescript II SK™ (Stratagene)or pUC18 (BRL; Gaithersburg Md.) for sequencing.

Sequencing was performed as described above, except that primers wereselected based on the cryptdin 1 cDNA sequence and with the expectationthat mouse cryptdin genes would be structurally homologous to the rabbitMCP-1 and MCP-2 defensin genes (see Huttner et al., supra, 1994). DNAsequence data were analyzed using the programs of Staden (Biochem. Soc.Trans. 12:1005-1008 (1984) and the University of Wisconsin GeneticsComputer Group (Devereux et al., Nucl. Acids Res, 12:387-395 (1985)).Searches for homology were performed as described above.

As shown in FIG. 11, screening of the genomic library produced nucleicacid sequences that contained the complete coding sequences for mousecryptdins 1, 2, 3, 5 and 6 (SEQ ID NOS: 58-62). In addition, ahomologous gene, designated cryptdin i (Crypi; SEQ ID NO: 63), whichapparently was inactivated due to a point mutation that changed acysteine codon to an in-frame stop codon, was isolated. Examination ofthe nucleic acid sequences revealed that the cryptdin genes contain twoexons, the first of which codes for the 5'-UTS and the prepro-codingregion and the second of which encodes the mature cryptdin peptide andthe 3'-UTS (not shown; but see FIG. 11.A.). A similar structure has beendescribed for the human cryptdin genes (Jones and Bevins, J. Biol. Chem.267:23216-23225 (1992)).

Similar methods as described above were used to obtain the cDNAsequences encoding rat cryptdins 1-3 (FIGS. 14.A. to 14.C.; SEQ ID NOS:65-67, respectively), except that RNA was obtained from the smallintestine of Sprague-Dawley rats. In addition, genomic DNA sequencesencoding rat cryptdins 1-3 (FIGS. 15.A. to 15.C.; SEQ ID NOS: 68-70,respectively) were obtained using methods as described above, exceptthat a genomic library containing Sprague-Dawley DNA cloned in EMBL3 waspurchased from Clontech (Palo Alto Calif.).

Although the invention has been described with reference to thedisclosed embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 70                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Leu Ser Lys Lys                                                          1                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Gly Ile Arg Phe Leu Tyr                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Arg Asn Leu Phe Leu Thr Phe Val Phe                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Arg Arg Gly His Leu Met Tyr Thr Leu                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: - #/note= "Xaa = Amino acid is                             independentl - #y L or M."                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 9                                                               (D) OTHER INFORMATION: - #/note= "Xaa = Amino acid is                              independentl - #y L or M"                                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Arg Lys Gly His Leu Xaa Tyr Thr Xaa                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Leu Arg Asp Leu Val Cys Tyr Cys Arg Ser Ar - #g Gly Cys Lys Gly Arg      1               5   - #                10  - #                15               - - Glu Arg Met Asn Gly Thr Cys Arg Lys Gly Hi - #s Leu Leu Tyr Thr Leu                  20      - #            25      - #            30                   - - Cys Cys Arg                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Leu Arg Asp Leu Val Cys Tyr Cys Arg Thr Ar - #g Gly Cys Lys Arg Arg      1               5   - #                10  - #                15               - - Glu Arg Met Asn Gly Thr Cys Arg Lys Gly Hi - #s Leu Met Tyr Thr Leu                  20      - #            25      - #            30                   - - Cys Cys Arg                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Leu Arg Asp Leu Val Cys Tyr Cys Arg Lys Ar - #g Gly Cys Lys Arg Arg      1               5   - #                10  - #                15               - - Glu Arg Met Asn Gly Thr Cys Arg Lys Gly Hi - #s Leu Met Tyr Thr Leu                  20      - #            25      - #            30                   - - Cys Cys Arg                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Gly Leu Leu Cys Tyr Cys Arg Lys Gly His Cy - #s Lys Arg Gly Glu Arg      1               5   - #                10  - #                15               - - Val Arg Gly Thr Cys Gly Ile Arg Phe Leu Ty - #r Cys Cys Pro Arg                      20      - #            25      - #            30                   - -  - - NFORMATION FOR SEQ ID NO:10:                                         - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Leu Ser Lys Lys Leu Ile Cys Tyr Cys Arg Il - #e Arg Gly Cys Lys Arg      1               5   - #                10  - #                15               - - Arg Glu Arg Val Phe Gly Thr Cys Arg Asn Le - #u Phe Leu Thr Phe Val                  20      - #            25      - #            30                   - - Phe Cys Cys                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Leu Lys Gln Cys His Cys Arg Lys Phe Cys Ar - #g Pro Tyr Glu Lys Ala      1               5   - #                10  - #                15               - - Glu Gly Ser Cys Arg Pro Gly Leu Phe Ile Ly - #s Arg Lys Ile Cys Cys                  20      - #            25      - #            30                   - - Ile Gln Gln Trp Thr Pro Gly                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Gly Leu Leu Cys Tyr Cys Arg Lys Gly His Cy - #s Lys Arg Gly Glu Arg      1               5   - #                10  - #                15               - - Val Arg Gly Thr Cys Gly Ile Arg Phe Leu Ty - #r Cys Cys Pro Arg Arg                  20      - #            25      - #            30                   - -  - - NFORMATION FOR SEQ ID NO:13:                                         - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Leu Ser Lys Lys Leu Ile Cys Tyr Cys Arg Il - #e Arg Gly Cys Lys Arg      1               5   - #                10  - #                15               - - Arg Glu Arg Val Phe Gly Thr Cys Arg Asn Le - #u Phe Leu Thr Phe Val                  20      - #            25      - #            30                   - - Phe Cys Cys Ser                                                                  35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Leu Arg Asp Leu Val Cys Tyr Cys Arg Ala Ar - #g Gly Cys Lys Gly Arg      1               5   - #                10  - #                15               - - Glu Arg Met Asn Gly Thr Cys Arg Lys Gly Hi - #s Leu Leu Tyr Met Leu                  20      - #            25      - #            30                   - - Cys Cys Arg                                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Leu Lys Gln Cys His Cys Arg Lys Phe Cys Ar - #g Pro Tyr Glu Lys Ala      1               5   - #                10  - #                15               - - Glu Gly Ser Cys Arg Pro Gly Leu Phe Ile Ly - #s Arg Lys Ile Cys Cys                  20      - #            25      - #            30                   - - Ile Gln Gln Trp Thr Pro Gly Arg Thr                                              35          - #        40                                              - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Ile Gly Arg Pro Val Arg Arg Cys Arg Cys Ar - #g Ala Asn Cys Gly Pro      1               5   - #                10  - #                15               - - Lys Glu Tyr Ala Thr Ala Phe Cys Ala Gln Gl - #y Pro Phe Lys Gln Phe                  20      - #            25      - #            30                   - - Lys Phe Cys Cys Thr                                                              35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Ile Arg Trp Pro Trp Lys Arg Cys His Cys Ar - #g Ser Phe Cys Arg Pro      1               5   - #                10  - #                15               - - Tyr Glu Asn Ala Thr Ser Phe Cys Ala Gln Gl - #y Leu Phe Lys Gln His                  20      - #            25      - #            30                   - - Lys Phe Cys Cys Leu Asp Thr Trp Pro Pro Ar - #g Met Lys                          35          - #        40          - #        45                       - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Thr Ser Gly Ser Gln Ala Arg Ala Thr Cys Ty - #r Cys Arg Thr Gly Arg      1               5   - #                10  - #                15               - - Cys Ala Thr Arg Glu Ser Leu Ser Gly Val Cy - #s Glu Ile Ser Gly Arg                  20      - #            25      - #            30                   - - Leu Tyr Arg Leu Cys Cys Arg                                                      35                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Ala Phe Thr Cys His Cys Arg Arg Ser Cys Ty - #r Ser Thr Glu Tyr Ser      1               5   - #                10  - #                15               - - Tyr Gly Thr Cys Thr Val Met Gly Ile Asn Hi - #s Arg Phe Cys Cys Leu                  20      - #            25      - #            30                   - -  - - NFORMATION FOR SEQ ID NO:20:                                         - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 103 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Met Lys Thr Leu Val Leu Leu Ser Ala Leu Va - #l Leu Leu Ala Phe Gln      1               5   - #                10  - #                15               - - Val Gln Ala Asp Pro Ile Gln Glu Ala Glu Gl - #u Glu Thr Lys Thr Glu                  20      - #            25      - #            30                   - - Glu Gln Pro Ala Asp Glu Asp Gln Asp Val Se - #r Val Ser Phe Glu Gly              35          - #        40          - #        45                       - - Pro Glu Pro Ser Ala Leu Gln Asn Leu Glu Il - #e Gly Trp Pro Leu Lys          50              - #    55              - #    60                           - - Gln Cys His Cys Arg Lys Phe Cys Arg Pro Ty - #r Glu Lys Ala Glu Gly      65                  - #70                  - #75                  - #80        - - Ser Cys Arg Pro Gly Leu Phe Ile Lys Arg Ly - #s Ile Cys Cys Ile Gln                      85  - #                90  - #                95               - - Gln Trp Thr Pro Gly Arg Thr                                                          100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Met Lys Thr Leu Val Leu Leu Ser Ala Leu Va - #l Leu Val Ala Tyr Gln      1               5   - #                10  - #                15               - - Val Gln Ala Asp Pro Ile Gln Gly Ala Glu Gl - #u Glu Thr Lys Thr Glu                  20      - #            25      - #            30                   - - Glu Gln Pro Ser Asp Glu Asp Gln Asp Val Se - #r Val Ser Phe Glu Gly              35          - #        40          - #        45                       - - Pro Glu Ala Ser Ala Leu Gln Asp Phe Glu Il - #e Gly Arg Pro Val Arg          50              - #    55              - #    60                           - - Arg Cys Arg Cys Arg Ala Asn Cys Gly Pro Ly - #s Glu Tyr Ala Thr Ala      65                  - #70                  - #75                  - #80        - - Phe Cys Ala Gln Gly Pro Phe Lys Gln Phe Ly - #s Arg Phe Cys Cys Thr                      85  - #                90  - #                95               - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 103 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Met Lys Thr Leu Val Leu Leu Ser Ala Leu Va - #l Leu Leu Ala Phe Gln      1               5   - #                10  - #                15               - - Ile Gln Ala Asp Pro Ile Gln Glu Ala Glu Gl - #u Glu Thr Lys Thr Glu                  20      - #            25      - #            30                   - - Glu Gln Pro Ala Asp Glu Asp Gln Asp Val Se - #r Val Ser Phe Glu Gly              35          - #        40          - #        45                       - - Pro Glu Pro Ser Ala Leu Gln Asn Leu Glu Il - #e Arg Trp Pro Trp Lys          50              - #    55              - #    60                           - - Arg Cys His Cys Arg Ser Phe Cys Arg Pro Ty - #r Glu Asn Ala Thr Ser      65                  - #70                  - #75                  - #80        - - Phe Cys Ala Gln Gly Leu Phe Lys Gln His Ly - #s Phe Cys Cys Leu Asp                      85  - #                90  - #                95               - - Thr Trp Pro Pro Arg Met Lys                                                          100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Met Lys Thr Leu Val Leu Leu Ser Ala Leu Va - #l Leu Leu Ala Phe Gln      1               5   - #                10  - #                15               - - Val Gln Ala Asp Pro Ile Gln Asn Thr Asp Gl - #u Glu Thr Lys Thr Glu                  20      - #            25      - #            30                   - - Glu Gln Pro Gly Glu Asp Asp Gln Ala Val Se - #r Val Ser Phe Gly Asp              35          - #        40          - #        45                       - - Pro Glu Gly Thr Ser Leu Gln Glu Glu Ser Le - #u Arg Asp Leu Val Cys          50              - #    55              - #    60                           - - Tyr Cys Arg Ser Arg Gly Cys Lys Gly Arg Gl - #u Arg Met Asn Gly Thr      65                  - #70                  - #75                  - #80        - - Cys Arg Lys Gly His Leu Leu Tyr Thr Leu Cy - #s Cys Arg                                  85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Met Lys Thr Leu Ile Leu Leu Ser Ala Leu Va - #l Leu Leu Ala Phe Gln      1               5   - #                10  - #                15               - - Val Gln Ala Asp Pro Ile Gln Asn Thr Asp Gl - #u Glu Thr Lys Thr Glu                  20      - #            25      - #            30                   - - Lys Gln Pro Gly Glu Glu Asp Gln Ala Val Se - #r Val Ser Phe Gly Asp              35          - #        40          - #        45                       - - Pro Glu Gly Ser Ser Leu Gln Glu Glu Ser Le - #u Arg Asp Leu Val Cys          50              - #    55              - #    60                           - - Tyr Cys Arg Thr Arg Gly Cys Lys Arg Arg Gl - #u Arg Met Asn Gly Thr      65                  - #70                  - #75                  - #80        - - Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cy - #s Cys Arg                                  85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Met Lys Thr Leu Val Leu Leu Ser Ala Leu Va - #l Leu Leu Ala Phe Gln      1               5   - #                10  - #                15               - - Val Gln Ala Asp Pro Ile Gln Asn Thr Asp Gl - #u Glu Thr Lys Thr Glu                  20      - #            25      - #            30                   - - Glu Gln Pro Gly Glu Asp Asp Gln Ala Val Se - #r Val Ser Phe Gly Asp              35          - #        40          - #        45                       - - Pro Glu Gly Ser Ser Leu Gln Glu Glu Ser Le - #u Arg Asp Leu Val Cys          50              - #    55              - #    60                           - - Tyr Cys Arg Lys Arg Gly Cys Lys Arg Arg Gl - #u Arg Met Asn Gly Thr      65                  - #70                  - #75                  - #80        - - Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cy - #s Cys Arg                                  85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 92 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 79                                                              (D) OTHER INFORMATION: - #/note= "Xaa = Amino acid is                              independentl - #y L or M."                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 80                                                              (D) OTHER INFORMATION: - #/note= "Xaa = Amino acid is                              independentl - #y L or M."                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 82                                                              (D) OTHER INFORMATION: - #/note= "Xaa = Amino acid is                              independentl - #y L or M."                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - Leu Val Leu Leu Ser Ala Leu Val Leu Leu Al - #a Phe Gln Val Gln Ala      1               5   - #                10  - #                15               - - Asp Pro Ile Gln Asn Thr Asp Glu Glu Thr Ly - #s Thr Glu Glu Gln Pro                  20      - #            25      - #            30                   - - Gly Glu Glu Asp Gln Ala Val Ser Ile Ser Ph - #e Gly Gly Gln Glu Gly              35          - #        40          - #        45                       - - Ser Ala Leu His Glu Lys Ser Leu Arg Gly Le - #u Leu Cys Tyr Cys Arg          50              - #    55              - #    60                           - - Lys Gly His Cys Lys Arg Gly Glu Arg Val Ar - #g Gly Thr Cys Xaa Xaa      65                  - #70                  - #75                  - #80        - - Gly Xaa Ile Arg Phe Leu Tyr Cys Cys Pro Ar - #g Arg                                      85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - Met Lys Thr Phe Val Leu Leu Ser Ala Leu Va - #l Leu Leu Ala Phe Gln      1               5   - #                10  - #                15               - - Val Gln Ala Asp Pro Ile His Lys Thr Asp Gl - #u Glu Thr Asn Thr Glu                  20      - #            25      - #            30                   - - Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Se - #r Ile Ser Phe Gly Gly              35          - #        40          - #        45                       - - Gln Glu Gly Ser Ala Leu His Glu Glu Leu Se - #r Lys Lys Leu Ile Cys          50              - #    55              - #    60                           - - Tyr Cys Arg Ile Arg Gly Cys Lys Arg Arg Gl - #u Arg Val Phe Gly Thr      65                  - #70                  - #75                  - #80        - - Cys Arg Asn Leu Phe Leu Thr Phe Val Phe Cy - #s Cys Ser                                  85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - Met Lys Thr Leu Val Leu Leu Ser Ala Leu Va - #l Leu Leu Ala Phe Gln      1               5   - #                10  - #                15               - - Val Gln Ala Asp Pro Ile Gln Asn Thr Asp Gl - #u Glu Thr Lys Thr Glu                  20      - #            25      - #            30                   - - Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Se - #r Val Ser Phe Gly Asp              35          - #        40          - #        45                       - - Pro Glu Gly Thr Ser Leu Gln Glu Glu Ser Le - #u Arg Asp Leu Val Cys          50              - #    55              - #    60                           - - Tyr Cys Arg Ala Arg Gly Cys Lys Gly Arg Gl - #u Arg Met Asn Gly Thr      65                  - #70                  - #75                  - #80        - - Cys Arg Lys Gly His Leu Leu Tyr Met Leu Cy - #s Cys Arg                                  85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - Met Lys Thr Leu Ile Leu Leu Ser Ala Leu Va - #l Leu Leu Ala Phe Gln      1               5   - #                10  - #                15               - - Val Gln Ala Asp Pro Ile Gln Asn Thr Asp Gl - #u Glu Thr Lys Thr Glu                  20      - #            25      - #            30                   - - Glu Gln Pro Gly Glu Asp Asp Gln Ala Val Se - #r Val Ser Phe Gly Asp              35          - #        40          - #        45                       - - Pro Glu Gly Ser Ser Leu Gln Glu Glu Ser Le - #u Arg Asp Leu Val Cys          50              - #    55              - #    60                           - - Tyr Cys Arg Thr Arg Gly Cys Lys Arg Arg Gl - #u His Met Asn Gly Thr      65                  - #70                  - #75                  - #80        - - Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cy - #s Cys Arg                                  85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - Met Lys Thr Leu Val Leu Leu Ser Ala Leu Va - #l Leu Leu Ala Phe Gln      1               5   - #                10  - #                15               - - Val Gln Ala Asp Pro Ile Gln Asn Thr Asp Gl - #u Glu Thr Lys Thr Glu                  20      - #            25      - #            30                   - - Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Se - #r Val Ser Phe Gly Asp              35          - #        40          - #        45                       - - Pro Glu Gly Ser Ser Leu Gln Glu Glu Ser Le - #u Arg Asp Leu Val Cys          50              - #    55              - #    60                           - - Tyr Cys Arg Lys Arg Gly Cys Lys Arg Arg Gl - #u His Met Asn Gly Thr      65                  - #70                  - #75                  - #80        - - Cys Arg Lys Gly His Leu Leu Tyr Met Leu Cy - #s Cys Arg                                  85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - Leu Ala Phe Gln Val Gln Ala Asp Pro Ile Gl - #n Asn Thr Asp Glu Glu      1               5   - #                10  - #                15               - - Thr Lys Thr Glu Glu Gln Pro Gly Glu Asp As - #p Gln Ala Val Ser Val                  20      - #            25      - #            30                   - - Ser Phe Gly Asp Pro Glu Gly Ser Ser Leu Gl - #n Glu Glu Ser Leu Arg              35          - #        40          - #        45                       - - Asp Leu Val Cys Tyr Cys Arg Lys Arg Gly Cy - #s Lys Arg Arg Glu His          50              - #    55              - #    60                           - - Met Asn Gly Thr Cys Arg Lys Gly His Leu Me - #t Tyr Thr Leu Cys Cys      65                  - #70                  - #75                  - #80        - - Arg                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 92 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - Lys Thr Leu Val Leu Leu Ser Ala Leu Val Le - #u Leu Ala Phe Gln Val      1               5   - #                10  - #                15               - - Gln Ala Asp Pro Ile Gln Asn Thr Asp Glu Gl - #u Thr Lys Thr Glu Glu                  20      - #            25      - #            30                   - - Gln Pro Gly Glu Asp Asp Gln Ala Val Ser Va - #l Ser Phe Gly Asp Pro              35          - #        40          - #        45                       - - Glu Gly Ser Ser Leu Gln Glu Glu Ser Leu Ar - #g Asp Leu Val Cys Tyr          50              - #    55              - #    60                           - - Cys Arg Lys Arg Gly Cys Lys Gly Arg Glu Ar - #g Met Asn Gly Thr Cys      65                  - #70                  - #75                  - #80        - - Arg Lys Gly His Leu Leu Tyr Thr Leu Cys Cy - #s Arg                                      85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 85 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - Ala Leu Val Leu Leu Ala Phe Gln Val Gln Al - #a Asp Pro Ile Gln Asn      1               5   - #                10  - #                15               - - Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pr - #o Gly Glu Glu Asp Gln                  20      - #            25      - #            30                   - - Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gl - #y Thr Ser Leu Gln Glu              35          - #        40          - #        45                       - - Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Ar - #g Ser Arg Gly Cys Lys          50              - #    55              - #    60                           - - Gly Arg Glu Arg Met Asn Gly Thr Cys Arg Ly - #s Gly His Leu Leu Tyr      65                  - #70                  - #75                  - #80        - - Met Leu Cys Cys Arg                                                                      85                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - Met Lys Thr Leu Ile Leu Leu Ser Ala Leu Va - #l Leu Leu Ala Phe Gln      1               5   - #                10  - #                15               - - Val Gln Ala Asp Pro Ile Gln Asn Thr Asp Gl - #u Glu Thr Lys Thr Glu                  20      - #            25      - #            30                   - - Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Se - #r Val Ser Phe Gly Asp              35          - #        40          - #        45                       - - Pro Glu Gly Thr Ser Leu Gln Glu Glu Ser Le - #u Arg Asp Leu Val Cys          50              - #    55              - #    60                           - - Tyr Cys Arg Ala Arg Gly Cys Lys Gly Arg Gl - #u Arg Met Asn Gly Thr      65                  - #70                  - #75                  - #80        - - Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cy - #s Cys Arg                                  85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - Met Lys Thr Leu Val Leu Leu Ser Ala Leu Va - #l Leu Leu Ala Phe Gln      1               5   - #                10  - #                15               - - Val Gln Ala Asp Pro Ile Gln Asn Thr Asp Gl - #u Glu Thr Lys Thr Glu                  20      - #            25      - #            30                   - - Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Se - #r Val Ser Phe Gly Asp              35          - #        40          - #        45                       - - Pro Glu Gly Thr Ser Leu Gln Glu Glu Ser Le - #u Arg Asp Leu Val Cys          50              - #    55              - #    60                           - - Tyr Cys Arg Lys Arg Gly Cys Lys Arg Arg Gl - #u His Met Asn Gly Thr      65                  - #70                  - #75                  - #80        - - Cys Arg Arg Gly His Leu Met Tyr Thr Leu Cy - #s Cys Arg                                  85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 85 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - Ala Leu Val Leu Leu Ala Phe Gln Val Gln Al - #a Asp Pro Ile Gln Asn      1               5   - #                10  - #                15               - - Thr Asp Glu Glu Thr Lys Thr Glu Glu Gln Pr - #o Gly Glu Glu Asp Gln                  20      - #            25      - #            30                   - - Ala Val Ser Val Ser Phe Gly Asp Pro Glu Gl - #y Ser Ser Leu Gln Glu              35          - #        40          - #        45                       - - Glu Ser Leu Arg Asp Leu Val Cys Tyr Cys Ar - #g Thr Arg Gly Cys Lys          50              - #    55              - #    60                           - - Arg Arg Glu Arg Met Asn Gly Thr Cys Arg Ly - #s Gly His Leu Met His      65                  - #70                  - #75                  - #80        - - Thr Leu Cys Cys Arg                                                                      85                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - Met Lys Thr Leu Val Leu Leu Ser Ala Leu Va - #l Leu Leu Ala Phe Gln      1               5   - #                10  - #                15               - - Val Gln Ala Asp Pro Ile Gln Asn Thr Asp Gl - #u Glu Thr Lys Thr Glu                  20      - #            25      - #            30                   - - Glu Gln Pro Gly Glu Asp Asp Gln Ala Val Se - #r Val Ser Phe Gly Asp              35          - #        40          - #        45                       - - Pro Glu Gly Ser Ser Leu Gln Glu Glu Ser Le - #u Arg Asp Leu Val Cys          50              - #    55              - #    60                           - - Tyr Cys Arg Lys Arg Gly Cys Lys Arg Arg Gl - #u His Ile Asn Gly Thr      65                  - #70                  - #75                  - #80        - - Cys Arg Lys Gly His Leu Leu Tyr Met Leu Cy - #s Cys Arg                                  85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - Met Lys Thr Leu Ile Leu Leu Ser Ala Leu Va - #l Leu Leu Ala Phe Gln      1               5   - #                10  - #                15               - - Val Gln Ala Asp Pro Ile Gln Asn Thr Asp Gl - #u Glu Thr Lys Thr Glu                  20      - #            25      - #            30                   - - Glu Gln Pro Gly Glu Glu Asp Gln Ala Val Se - #r Val Ser Phe Gly Asp              35          - #        40          - #        45                       - - Pro Glu Gly Thr Ser Leu Gln Glu Glu Ser Le - #u Arg Asp Leu Val Cys          50              - #    55              - #    60                           - - Tyr Cys Arg Ser Arg Gly Cys Lys Gly Arg Gl - #u Arg Met Asn Gly Thr      65                  - #70                  - #75                  - #80        - - Cys Arg Lys Gly His Leu Met Tyr Thr Leu Cy - #s Cys Arg                                  85  - #                90                                      - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 82 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - Leu Leu Ala Phe Gln Val Gln Ala Asp Pro Il - #e Gln Asn Thr Asp Glu      1               5   - #                10  - #                15               - - Glu Thr Lys Thr Glu Glu Gln Pro Gly Glu Gl - #u Asp Gln Ala Val Ser                  20      - #            25      - #            30                   - - Val Ser Phe Gly Asp Pro Glu Gly Thr Ser Le - #u Gln Glu Glu Ser Leu              35          - #        40          - #        45                       - - Arg Asp Leu Val Cys Tyr Cys Arg Lys Arg Gl - #y Cys Lys Arg Arg Glu          50              - #    55              - #    60                           - - His Met Asn Gly Thr Cys Arg Lys Gly His Le - #u Leu Tyr Thr Leu Cys      65                  - #70                  - #75                  - #80        - - Cys Arg                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 422 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - - ACACATTGAG CTCCTGCTCA CCAATCCTCC AGGTGACTCC CAGCCATGAA GA -             #CACTAGTC     60                                                                 - - CTCCTCTCTG CCCTTGTCCT GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CC -            #AAAACACA    120                                                                 - - GATGAAGAGA CTAAAACTGA GGAGCAGCCA GGGGAAGACG ACCAGGCCGT AT -            #CTGTCTCC    180                                                                 - - TTTGGAGACC CAGAAGGCAC TTCTCTTCAA GAGGAATCGT TGAGAGATCT GG -            #TATGCTAT    240                                                                 - - TGTAGATCAA GAGGCTGCAA AGGAAGAGAA CGCATGAATG GGACCTGCAG AA -            #AGGGTCAT    300                                                                 - - TTATTGTACA CGCTCTGCTG TCGCTGAACA TGGAGACCAC AGAGGACAAG AC -            #GAACATGA    360                                                                 - - GTACTGAGGC CACTGATGCT GGTGCCTGAT GACCACTTCG CAATAAATTG TT -            #CGCAATAT    420                                                                 - - GC                  - #                  - #                  - #                 422                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 422 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - ACACACTGAG CCGCTACTCA CCAATCCTCC AGGTGACTCC CAGCCATGAA GA -             #CACTAATC     60                                                                 - - CTCCTCTCTG CCCTCGTCCT GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CC -            #AAAATACA    120                                                                 - - GATGAAGAGA CTAAAACTGA GAAGCAGCCA GGGGAAGAGG ACCAGGCCGT AT -            #CTGTCTCC    180                                                                 - - TTTGGAGACC CAGAAGGCTC TTCTCTTCAA GAGGAATCGT TGAGAGATCT GG -            #TATGCTAT    240                                                                 - - TGTAGAACAA GAGGCTGCAA AAGAAGAGAA CGCATGAATG GGACCTGCAG AA -            #AGGGTCAT    300                                                                 - - TTAATGTACA CGCTCTGCTG TCGCTGAACA TGGAGACCAC AGAGGACAAG AT -            #GACCATGA    360                                                                 - - GTACTGAGGC CACTGATGCT GGTGCCTGAT GACCACTTCG CAATAAATTG CT -            #TGCAATAT    420                                                                 - - GC                  - #                  - #                  - #                 422                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 422 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - - ACACATTGGG CTCCTGCTCA CCAATCCTCC AGGTGACTCC CAGCCATGAA GA -             #CACTAGTC     60                                                                 - - CTCCTCTCTG CCCTCGTCCT GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CC -            #AAAACACA    120                                                                 - - GATGAAGAGA CTAAAACTGA GGAGCAGCCA GGGGAAGACG ACCAGGCCGT AT -            #CTGTCTCC    180                                                                 - - TTTGGAGACC CAGAAGGCTC TTCTCTTCAA GAGGAATCGT TGAGAGATCT GG -            #TATGCTAT    240                                                                 - - TGTAGAAAAA GAGGCTGCAA AAGAAGAGAA CGCATGAATG GGACCTGCAG AA -            #AGGGTCAT    300                                                                 - - TTAATGTACA CACTCTGCTG TCGCTGAACA TGGAGACCAC AGAGGACAAG AC -            #GAACATGA    360                                                                 - - GTACTGAGGC CACTGATGCT GGTGCCTGAT GACCACTTCG CAATAAATTG TT -            #CGCAATAT    420                                                                 - - GC                  - #                  - #                  - #                 422                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 365 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - - ACTAGTCCTC CTCTCTGCCC TCGTCCTGCT GGCCTTCCAG GTCCAGGCTG AT -             #CCTATCCA     60                                                                 - - AAATACAGAT GAAGAGACTA AAACTGAGGA GCAGCCAGGG GAAGAGGACC AG -            #GCCGTATC    120                                                                 - - TGTCTCCTTT GGAGACCCAG AAGGCTCTGC TCTTCATGAA AAATCTTTGA GA -            #GGTTTGTT    180                                                                 - - ATGCTATTGT AGAAAAGGAC ACTGCAAAAG AGGAGAACGA GTTCGTGGGA CT -            #TGTGGAAT    240                                                                 - - ACGATTTTTG TACTGCTGCC CCCGCCGCTG AACATGCAGA TGACAAAGAT AT -            #GACAACCA    300                                                                 - - TTGTCTCTGA GGCCGCTGAT GCCGGGGCCT GATGACCACT TCTCAAGAAA TG -            #TTTGCAAT    360                                                                 - - ATGCA                 - #                  - #                  -      #           365                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 421 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - - ACACATTGGG CTCCTGCTCA CCAATTCTCC AGGTGACCCC CAGCCATGAA GA -            #CATTTGTC     60                                                                 - - CTCCTCTCTG CCCTTGTCCT GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CC -            #ACAAAACA    120                                                                 - - GATGAAGAGA CTAATACTGA GGAGCAGCCA GGGGAAGAGG ACCAGGCCGT AT -            #CTATCTCC    180                                                                 - - TTTGGAGGCC AAGAAGGGTC TGCTCTTCAT GAGGAATTGT CAAAAAAGCT GA -            #TATGCTAT    240                                                                 - - TGTAGAATAA GAGGCTGCAA AAGAAGAGAA CGCGTTTTTG GGACCTGCAG AA -            #ATCTTTTT    300                                                                 - - TTAACTTTCG TATTCTGCTG CAGCTGAATA TGCAGATGAC AAAGATATGA CA -            #ACCATCAG    360                                                                 - - CACTGAGGCC ACTGATGCTG GGGTCTGATG ATCACCTCGC AATAAATTGT TC -            #GCAATATG    420                                                                 - - C                  - #                  - #                  - #                  421                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 422 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - - ACACACTGAG CTGCTACTCA CCAATCCTCC AGGTGACTCC CAGCCATGAA GA -             #CACTAATC     60                                                                 - - CTCCTCTCTG CCCTCGTCCT GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CC -            #AAAATACA    120                                                                 - - GATGAAGAGA CTAAAACTGA GGAGCAGCCA GGGGAAGAGG ACCAGGCCGT AT -            #CTGTCTCC    180                                                                 - - TTTGGAGACC CAGAAGGCAC TTCTCTTCAA GAGGAATCAT TGAGATATCT GG -            #TATGCTAT    240                                                                 - - TGTAGAGCAA GAGGCTGCAA AGGAAGAGAA CGCATGAATG GGACCTGCAG AA -            #AGGGTCAT    300                                                                 - - TTATTGTACA TGCTCTGCTG TCGCTGAACA TGGAGACCTC AGAGAACAAG AC -            #GACCATGA    360                                                                 - - GTACTGAGGC CACTGATGCT GGTGCCTGAT GACCACTTCG CAATACATTG TT -            #CGCAATAT    420                                                                 - - GC                  - #                  - #                  - #                 422                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 420 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                              - - ACACTGAGCT GCTACTCACC AATCCTCCAG GTGACTCCCA GCCATGAAGA CA -             #CTAATCCT     60                                                                 - - CCTCTCTGCC CTCGTCCTGC TGGCCTTCCA GGTCCAGGCT GATCCTATCC AA -            #AACACAGA    120                                                                 - - TGAAGAGACT AAAACTGAGG AGCAGCCAGG GGAAGACGAC CAGGCCGTAT CT -            #GTCTCCTT    180                                                                 - - TGGAGACCCA GAAGGCTCTT CTCTTCAAGA GGAATCGTTG AGAGATCTGG TA -            #TGCTATTG    240                                                                 - - TAGAACAAGA GGCTGCAAAA GAAGAGAACA CATGAATGGG ACCTGCAGAA AG -            #GGTCATTT    300                                                                 - - AATGTACACG CTCTGCTGTC GCTGAACATG GAGACCTCAG AGAACAAGAC GA -            #CCATGAGT    360                                                                 - - ACTGAGGCCA CTGATGCTGG TGCCTGATGA CCACTTCGCA ATAAATTGTT CG -            #CAATATGC    420                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 342 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                              - - GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CCAAAACACA GATGAAGAGA CT -            #AAAACTGA     60                                                                 - - GGAGCAGCCA GGGGAAGACG ACCAGGCCGT ATCTGTCTCC TTTGGAGACC CA -            #GAAGGCTC    120                                                                 - - TTCTCTTCAA GAGGAATCGT TGAGAGATCT GGTATGCTAT TGTAGAAAAA GA -            #GGCTGCAA    180                                                                 - - AAGAAGAGAA CACATGAATG GGACCTGCAG AAAGGGTCAT TTAATGTACA CG -            #CTCTGCTG    240                                                                 - - TCGCTGAACA TGGAGACCAC AGAGGACAAG ACAAGCATGA GTACTGAGGC CA -            #CTGATGCT    300                                                                 - - GGTGCCTGAT GACCACTTCG CAATAAATTG TTCGCAATAT GC    - #                      - # 342                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 377 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                              - - ATGAAGACAC TAGTCCTCCT CTCTGCCCTC GTCCTGCTGG CCTTCCAGGT CC -             #AGGCTGAT     60                                                                 - - CCTATCCAAA ACACAGATGA AGAGACTAAA ACTGAGGAGC AGCCAGGGGA AG -            #AGGACCAG    120                                                                 - - GCCGTATCTG TCTCCTTTGG AGACCCAGAA GGCTCTTCTC TTCAAGAGGA AT -            #CGTTGAGA    180                                                                 - - GATCTGGTAT GCTATTGTAG AAAAAGAGGC TGCAAAAGAA GAGAACACAT GA -            #ATGGGACC    240                                                                 - - TGCAGAAAGG GTCATTTATT GTACATGCTC TGCTGTCGCT GAACATGGAG AC -            #CACAGAGG    300                                                                 - - ACAAGATGAA CATGAGTACT GAGGCCACTG ATGCTGGTGC CTGATGACCA CT -            #TCGCAATA    360                                                                 - - AATTGTTCGC AATATGC             - #                  - #                      - #  377                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 375 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                              - - GAAGACACTA GTCCTCCTCT CTGCCCTCGT CCTGCTGGCC TTCCAGGTCC AG -             #GCTGATCC     60                                                                 - - TATCCAAAAC ACAGATGAAG AGACTAAAAC TGAGGAGCAG CCAGGGGAAG AC -            #GACCAGGC    120                                                                 - - CGTATCTGTC TCCTTTGGAG ACCCAGAAGG CTCTTCTCTT CAAGAGGAAT CG -            #TTGAGAGA    180                                                                 - - TCTGGTATGC TATTGTAGAA AAAGAGGCTG CAAAGGAAGA GAACGCATGA AT -            #GGAACCTG    240                                                                 - - CAGAAAGGGT CATTTATTGT ACACGCTCTG CTGTCGCTGA ACATGGAGAC CA -            #CAGAGGAC    300                                                                 - - AAGACGAACA TGAGTACTGA GGCCACTGAT GCTGGTGCCT GATGACCACT TC -            #GCAATAAA    360                                                                 - - TTGTTCGCAA TATGC              - #                  - #                      - #   375                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 352 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                              - - CCCTCGTCCT GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CCAAAACACA GA -             #TGAAGAGA     60                                                                 - - CTAAAACTGA GGAGCAGCCA GGGGAAGAGG ACCAGGCCGT ATCTGTCTCC TT -            #TGGAGACC    120                                                                 - - CAGAAGGCAC TTCTCTTCAA GAGGAATCGT TGAGAGATCT GGTATGCTAT TG -            #TAGATCAA    180                                                                 - - GAGGCTGCAA AGGAAGAGAA CGCATGAATG GAACCTGCAG AAAGGGTCAT TT -            #ATTGTACA    240                                                                 - - TGCTCTGCTG TCGCTGAACA TGGAGACCAC AGAGAACAAG ACGACCATGA GT -            #ACTGAGGC    300                                                                 - - CACTGATGCT GGTGCCTGAT GACCACTTCG CAATACATTG TTCGCAATAT GC - #                352                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 422 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                              - - ACACATTGGG CTCCTGCTCA CCAATCCTCC AGGTGACTCC CAGCCATGAA GA -             #CACTAGTC     60                                                                 - - CTCCTCTCTG CCCTCGTCCT GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CC -            #AAAACACA    120                                                                 - - GATGAAGAGA CTAAAACTGA GGAGCAGCCA GGGGAAGAGG ACCAGGCCGT AT -            #CTGTCTCC    180                                                                 - - TTTGGAGACC CAGAAGGCAC TTCTCTCCAA GAGGAATAGT TGAGAGATCT GG -            #TATGCTAT    240                                                                 - - TGTAGAGCAA GAGGCTGCAA AGGAAGAGAA CGCATGAATG GGACCTGCAG AA -            #AGGGTCAT    300                                                                 - - TTAATGTACA CGCTCTGCTG TCGCTGAACA TGGAGACCTC AGAGAACAAG AC -            #GACCATGA    360                                                                 - - GTACTGAGGC CACTGATGCT GGTGCCTGAT GACCACTTCG CAATAAATTG TT -            #CGCAATAT    420                                                                 - - GC                  - #                  - #                  - #                 422                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 388 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                              - - GACTCCCAGC CATGAAGACA CTAGTCCTCC TCTCTGCCCT TGTCCTGCTG GC -             #CTTCCAGG     60                                                                 - - TCCAGGCTGA TCCTATCCAA AACACAGATG AAGAGACTAA AACTGAGGAG CA -            #GCCAGGAG    120                                                                 - - AAGAGGACCA GGCCGTATCT GTCTCCTTTG GAGACCCAGA AGGCACTTCT CT -            #TCAAGAGG    180                                                                 - - AATCGTTGAG AGATCTGGTA TGCTATTGTA GAAAAAGAGG CTGCAAAAGA AG -            #AGAACACA    240                                                                 - - TGAATGGGAC CTGCAGAAGG GGTCATTTAA TGTACACACT CTGCTGTCGC TG -            #AACATGGA    300                                                                 - - GACCACAGAG GACAAGACGA ACATGAGTAC TGAGGCCACT GATGCTGGTG CC -            #TGATGACC    360                                                                 - - ACCTCGCAAT AAATTGTTCG CAATATGC         - #                  - #                388                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 352 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                              - - CCCTCGTCCT GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CCAAAACACA GA -             #TGAAGAGA     60                                                                 - - CTAAAACTGA GGAGCAGCCA GGGGAAGAGG ACCAGGCCGT ATCTGTCTCC TT -            #TGGAGACC    120                                                                 - - CAGAAGGCTC TTCTCTTCAA GAGGAATCGT TGAGAGATCT GGTATGCTAT TG -            #TAGAACAA    180                                                                 - - GAGGCTGCAA AAGAAGAGAA CGCATGAATG GGACCTGCAG AAAGGGTCAT TT -            #AATGCACA    240                                                                 - - CGCTCTGCTG TCGCTGAACA TGGAGACCAC AGAGGACAAG ACGAGCATGA GT -            #ACTGAGGC    300                                                                 - - CACTGATGCT GGTGCCTGAT GACCACTTCG CAATAAATTG TTCGCAAAAT GC - #                352                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 401 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                              - - CCAATCCTCC CAGTGACTCC CAGCCATGAA GACACTAGTC CTCCTCTCTG CC -             #CTTGTCCT     60                                                                 - - GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CCAAAACACA GATGAAGAGA CT -            #AAAACTGA    120                                                                 - - GGAGCAGCCA GGGGAAGACG ACCAGGCCGT ATCTGTCTCC TTTGGAGACC CA -            #GAAGGCTC    180                                                                 - - TTCTCTTCAA GAGGAATCGT TGAGAGATCT GGTATGCTAT TGTAGAAAAA GA -            #GGCTGCAA    240                                                                 - - AAGAAGAGAA CACATAAATG GGACCTGCAG AAAGGGTCAT TTATTGTACA CT -            #CTCTGCTG    300                                                                 - - TCGCTGAACA TGGAGACCAC AGAGGACAAG ATGACCATGA GTACTGAGGC CA -            #CTGATGCT    360                                                                 - - GGTGCCTGAT GACCACTCGC AATAAATTGT TCGCAATATG C    - #                      - #  401                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 391 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                              - - GGTGACTCCC AGCCATGAAG ACACTAATCC TCCTCTCTGC CCTCGTCCTG CT -             #GGCCTTCC     60                                                                 - - AGGTCCAGGC TGATCCTATC CAAAACACAG ATGAAGAGAC TAAAACTGAG GA -            #GCAGCCAG    120                                                                 - - GAGAAGAGGA CCAGGCCGTA TCTGTCTCCT TTGGAGACCC AGAAGGCACT TC -            #TCTTCAAG    180                                                                 - - AGGAATCGTT GAGAGATCTG GTATGCTATT GTAGATCAAG AGGCTGCAAA GG -            #AAGAGAAC    240                                                                 - - GCATGAATGG GACCTGCAGA AAGGGTCATT TAATGTACAC GCTCTGCTGT CG -            #CTGAACAT    300                                                                 - - GGAGACCTCA GAGAACAAGA CGACCATGAG TACTGAGGCC ACTGATGCTG GT -            #GCCTGATG    360                                                                 - - ACCACTTCGC AATAAATTGT TCGCAATATG C        - #                  - #             391                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 342 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                              - - GCTGGCCTTC CAGGTCCAGG CTGATCCTAT CCAAAATACA GATGAAGAGA CT -             #AAAACTGA     60                                                                 - - GGAGCAGCCA GGAGAAGAGG ACCAGGCCGT ATCTGTCTCC TTTGGAGACC CA -            #GAAGGCAC    120                                                                 - - TTCTCTTCAA GAGGAATCGT TGAGAGATCT GGTATGCTAT TGTAGAAAAA GA -            #GGCTGCAA    180                                                                 - - AAGAAGAGAA CACATGAATG GGACCTGCAG AAAGGGTCAT TTATTGTACA CG -            #CTCTGCTG    240                                                                 - - TCGCTGAACA TGGAGACCAC AGAGGACAAG ATGACCATGA GTACTGAGGC CA -            #CTGATGCT    300                                                                 - - GGTGCCTGAT GACCACCTCG CAATAAATTG CTTGCAATAT GC    - #                      - # 342                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 403 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                              - - ACACATGGCT CTCTCACCAA TCCTCCAGGT GACTCCCAGC CATGAAGACA CT -             #AGTCCTCC     60                                                                 - - TCTCTGCCTG TCCTGCTGGC CTTCCAGGTC CAGGCTGATC CTATCCAAAA CA -            #CAGATGAA    120                                                                 - - GAGACTAAAA CTGAGGAGCA GCCAGGGGAA GAGACCAGGC TGTGTCTGTC TC -            #TTTTGGAG    180                                                                 - - ACCCAGAAGG CCTTCTCTTC AAGAGGAATC GTTGAGAGAT CTGGTATGCT AT -            #TGTAGAAA    240                                                                 - - GAGGCTGCAA AGAAGAGAAC CATGAATGGG ACCTGCAGAA AGGGTCATTT AT -            #GTACAGCT    300                                                                 - - CTGCTGTCGC TGAACATGGA GACCCAGAGA CAAGAACATG AGTACTGAGG CC -            #ACTGATGC    360                                                                 - - TGGTGCCTGA TGACCACTTC TCAATAAATT GTTCGCAATA TGC    - #                      - #403                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 419 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 279..286                                                        (D) OTHER INFORMATION: - #/note= "N represents DNA that was                        not seque - #nced."                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                              - - TATAAATGCA GGCTGGATAT TCACTGTCCA CACATTGAGC TCCTGCTCAC CA -             #ATCCTCCA     60                                                                 - - GGTGACTCCC AGCCATGAAG ACACTAGTCC TCCTCTCTGC CCTTGTCCTG CT -            #GGCCTTCC    120                                                                 - - AGGTCCAGGC TGATCCTATC CAAAACACAG ATGAAGAGAC TAAAACTGAG GA -            #GCAGCCAG    180                                                                 - - GAGAAGAGGA CCAGGCCGTA TCTGTCTCCT TTGGAGACCC AGAAGGCACT TC -            #TCTTCAAG    240                                                                 - - AGGAATGTGA GTACTGGTGT CCAGAGTGAT GGATGCTTNN NNNNNNTTTT GT -            #ATCTCCAG    300                                                                 - - CGTTGAGAGA TCTGGTATGC TATTGTAGAT CAAGAGGCTG CAAAGGAAGA GA -            #ACGCATGA    360                                                                 - - ATGGAACCTG CAGAAAGGGT CATTTATTGT ACACGCTCTG CTGTCGCTGA AC -            #ATGGAGA     419                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 419 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 279..286                                                        (D) OTHER INFORMATION: - #/note= "N represents DNA that was                       not seque - #nced."                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                              - - TATAAATGCA GACTGGCTCC TCACTCTCCA CACATTGGGC TCCTGCTCAC CA -             #ATCCTCCC     60                                                                 - - AGTGACTCCC AGCCATGAAG CCACTTGTCC TCCTTTCTGC CCTTGTCCTA CT -            #GTCCTTTC    120                                                                 - - AGGTCCAGGC TGATCCTATC CAAAACACAG ATGAAGAGAC TAAAACTGAG GA -            #GCAGTCAG    180                                                                 - - GTGAAGAGGA CCAGGCTGTG TCTGTCTCCT TTGGAGACCG AGAAGGCGCT TC -            #TCTTCAAG    240                                                                 - - AAGAATGTGA GTACTGGTGC CCAGTGTGAT GGATGCTTNN NNNNNNTTTT GT -            #GTCTCCAG    300                                                                 - - CGTTGAGAGA TCTGGTATGC TATTGTAGAA CAAGAGGTTG CAAAAGAAGA GA -            #ACGCATGA    360                                                                 - - ATGGGACCTG CAGAAAGGGT CATTTAATGT ACACGCTCTG CTGCCGCTGA AC -            #ATGGAGA     419                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 419 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 279..286                                                        (D) OTHER INFORMATION: - #/note= "N represents DNA that was                       not seque - #nced."                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                              - - TATAAATGCA GGCTGGATAT TCACTCTCCA CACATTGGGC TCCTGCTCAC CA -             #ATCCTCCA     60                                                                 - - GGTGACTCCC AGCCATGAAG ACACTAGTCC TCCTCTCTGC CCTCGTCCTG CT -            #GGCCTTCC    120                                                                 - - AGGTCCAGGC TGATCCTATC CAAAACACAG ATGAAGAGAC TAAAACTGAG GA -            #GCAGCCAG    180                                                                 - - GGGAAGACGA CCAGGCTGTG TCTGTCTCTT TTGGAGACCC AGAAGGCTCT TC -            #TCTTCAAG    240                                                                 - - AGGAATGTGA GTATTGGTGT CCTGTGTGAT GGATGCTTNN NNNNNNTTTT GT -            #GTCTCCAG    300                                                                 - - CGTTGAGAGA TCTGGTATGC TATTGTAGAA AAAGAGGCTG CAAAAGAAGA GA -            #ACGCATGA    360                                                                 - - ATGGGACCTG CAGAAAGGGT CATTTAATGT ACACACTCTG CTGTCGCTGA AC -            #ATGGAGA     419                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 419 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 279..286                                                        (D) OTHER INFORMATION: - #/note= "N represents DNA that was                       not seque - #nced."                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                              - - TATAAATGCA AGTTGGCTAC TCACTCTCCA CACATTGGGC TCCTGCTCAC CA -             #ATTCTCCA     60                                                                 - - GGTGACCCCC AGCCATGAAG ACATTTGTCC TCCTCTCTGC CCTTGTCCTG CT -            #GGCCTTCC    120                                                                 - - AGGTCCAGGC TGATCCTATC CACAAAACAG ATGAAGAGAC TAATACTGAG GA -            #GCAGCCAG    180                                                                 - - GGGAAGAGGA CCAGGCTGTG TCAGTCTCCT TTGGAGGCCA AGAAGGGTCT GC -            #TCTTCATG    240                                                                 - - AAGAATGTGA GTAGTGGTAC GCAGTGTGAT GGATGCTTNN NNNNNNTTTT GT -            #GTCTCCAG    300                                                                 - - TGTCAAAAAA GCTGATATGC TATTGTAGAA TAAGAGGCTG CAAAAGAAGA GA -            #ACGCGTTT    360                                                                 - - TTGGGACCTG CAGAAATCTT TTTTTAACTT TCGTATTCTG CTGTAGCTGA AT -            #ATGCAGA     419                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 419 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 279..286                                                        (D) OTHER INFORMATION: - #/note= "N represents DNA that was                       not seque - #nced."                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                              - - TATAAATGCA GGCTGGATAT TCACTCTCCA CACACTGAGC TGCTACTCAC CA -             #ATCCTCCA     60                                                                 - - GGTGACTCCC AGCCATGAAG ACACTAATCC TCCTCTCTGC CCTCGTCCTG CT -            #GGCCTTCC    120                                                                 - - AGGTCCAGGC TGATCCTATC CAAAATACAG ATGAAGAGAC TAAAACTGAG GA -            #GCAGCCAG    180                                                                 - - GGGAAGAGGA CCAGGCTGTG TCTGTCTCTT TTGGAGACCC AGAAGGCACT TC -            #TCTTCAAG    240                                                                 - - AGGAATGTGA GTACTGGTGT CCAGTGTGAT GGATGCTTNN NNNNNNTTTT GT -            #GTCTCCAG    300                                                                 - - CATTGAGAGA TCTGGTATGC TATTGTAGAG CAAGAGGCTG CAAAGGAAGA GA -            #ACGCATGA    360                                                                 - - ATGGGACCTG CAGAAAGGGT CATTTATTGT ACATGCTCTG CTGTCGCTGA AC -            #ATGGAGA     419                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 419 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 279..286                                                        (D) OTHER INFORMATION: - #/note= "N represents DNA that was                       not seque - #nced."                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                              - - ACTTGAGGGT AACAGCCTCT CCCAATTCCA CACATTGAGC TCCTGCTCAC CA -             #ATCCTCCA     60                                                                 - - GGTGACTCCC AGCCATGAAG ACACTAGTCC TCCTCTCTGC CCTTGCCCTG CT -            #GGCCTTCC    120                                                                 - - AAGTCCAGGC TGATCCTATC CAAAACACAG ATGAAGAGAC TAAAACTGAG GA -            #GCAGCCAG    180                                                                 - - GGAAAGAAGA CCAAGCTGTT TCTGTCTCCT TTGGAGACCC AGAAGGCTCT TC -            #TCTTCAAG    240                                                                 - - AGGAATGTGA GTACTGGTGC CCAGTGTGAT GGATGCTTNN NNNNNNTTTT GT -            #GTCTCCAG    300                                                                 - - CGTTGAGAGA TCTGATATGA TATTGTAGAA CAAGAGGCTG CAAAAGAAGA GA -            #ACGCCTGA    360                                                                 - - ATGGGACCTG AAGAAAGGGT CATTTATTGT ACATGCTCTG CTGCTGCTGA AC -            #ATGGAGA     419                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 411 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                              - - TATAAATGCA RRCTGGMTMY TCACTNTCCA CACATTGRGC TCCTGCTCAC CA -            #ATCCTCCA     60                                                                 - - GGTGACTCCC AGCCATGAAG ACACTWGTCC TCCTCTCTGC CCTYGTCCTG CT -            #GGCCTTCC    120                                                                 - - AGGTCCAGGC TGATCCTATC CAAAAHACAG ATGAAGAGAC TAAAACTGAG GA -            #GCAGCCAG    180                                                                 - - GDGAAGARGA CCAGGCTGTD TCTGTCTCYT TTGGAGACCV AGAAGGCDCT TC -            #TCTTCAAG    240                                                                 - - ARGAATGTGA GTABTGGTGY CCAGTGTGAT GGATGCTTTT TTGTGTCTCC AG -            #CGTTGAGA    300                                                                 - - GATCTGRTAT GCTATTGTAG ADHAAGAGGC TGCAAARGAA GAGAACGCVT GA -            #ATGGGACC    360                                                                 - - TGCAGAAAGG GTCATTTAWT GTACANNCTC TGCTGYRGCT GAACATGGAG A - #                411                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 445 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                              - - ACACTGGTCT CCAGCTCACC AATCCTCCAG GTGACTTCCA GCCATGAAGA CT -             #CTTGTCCT     60                                                                 - - CCTCTCTGCC CTTGTCCTGC TGGCATTCCA GGTCCAGGCT GATCCCATTC AA -            #GAGGCAGA    120                                                                 - - AGAAGAGACT AAAACTGAGG AGCAGCCAGC AGATGAGGAC CAGGATGTGT CT -            #GTCTCCTT    180                                                                 - - TGAAGGCCCA GAACCCTCTG CTCTTCAAAA TTTAGAGATA GGATGGCCAT TA -            #AAGCAGTG    240                                                                 - - CCATTGCCGA AAGTTCTGCA GACCTTATGA AAAGGCCGAG GGGTCCTGTC GT -            #CCAGGTCT    300                                                                 - - ATTTATAAAA CGCAAAATCT GCTGCATACA ACAATGGACA CCAGGGAGGA CA -            #TAACCACG    360                                                                 - - TGAACTGGGA CCTCACAATC TGTCATTCTT GGGCTTCAAC TCGACTGCTT TT -            #CCTTCTCC    420                                                                 - - AATAAACCCC TTGCAGACAA AAAAA          - #                  - #                  445                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 445 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                              - - ACACTGGTCT CCAGCTCACC AATCCTCCAG GTGACTTCCA GCCATGAAGA CT -             #CTTGTCCT     60                                                                 - - CCTCTCTGCC CTTGTCCTGG TGGCCTACCA GGTCCAGGCT GATCCCATTC AA -            #GGGGCAGA    120                                                                 - - AGAAGAGACT AAAACTGAAG AGCAACCATC AGATGAGGAC CAGGATGTGT CT -            #GTCTCCTT    180                                                                 - - TGAAGGCCCA GAAGCCTCTG CTCTTCAAGA TTTTGAGATA GGAAGGCCAG TG -            #AGGAGGTG    240                                                                 - - CCGTTGCAGA GCAAACTGCG GACCTAAAGA ATATGCCACT GCGTTCTGTG CT -            #CAAGGTCC    300                                                                 - - ATTTAAACAG TTCAAATTCT GCTGCACATG AACATGGATC CCAAGTCTGA GA -            #TAACCACG    360                                                                 - - TGCTCTGGGA CCTCACAATC TGTCATTATT GTGCTTGACC TCAACTGCTT TT -            #CCTTCTCC    420                                                                 - - AATAAACTCC TGGCAGACAA AAAAA          - #                  - #                  445                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 445 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                              - - ACACTGGTCT CCAGCTCACC AATCCTCCAG GTGACTTCCA GCCATGAAGA CT -             #CTTGTCCT     60                                                                 - - CCTCTCTGCC CTTGTCCTGC TGGCATTCCA GATCCAGGCT GATCCCATTC AA -            #GAGGCAGA    120                                                                 - - AGAAGAGACT AAAACTGAGG AGCAGCCAGC AGATGAGGAC CAGGATGTGT CT -            #GTCTCCTT    180                                                                 - - TGAAGGCCCA GAACCCTCTG CTCTTCAAAA TTTAGAGATC AGATGGCCAT GG -            #AAGAGGTG    240                                                                 - - CCATTGCAGA AGTTTCTGCA GACCTTATGA AAATGCCACT TCGTTCTGTG CT -            #CAAGGTCT    300                                                                 - - ATTTAAACAA CACAAATTCT GCTGCCTAGA AACATGGCCC CCAAGGATGA AA -            #TAACCACG    360                                                                 - - TGCTCTGGGA CCTCACAATC TGTCATCATT GTGCTTGGCC TCAACTTCTT TT -            #CCTTCTCC    420                                                                 - - AATAAACTCC TTGCAGACAA AAAAA          - #                  - #                  445                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2457 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                              - - CCTGAGACCA ACTCTGTGAT AATCAGAAAA GTCAATAATG TGTCTGAAAT GT -             #AAGGTGTG     60                                                                 - - CTTCTTGACT GATAGTTCTA AGCCTACAGA GAGATTCATG TGGTCATATC CC -            #ATTTAACA    120                                                                 - - ATGATATATA TGTTAAATAT ATAAAGATAT ATGTATGTTC AGTATGTATG TT -            #CAATATGT    180                                                                 - - ATGTAAATAA TATTCTTGCT GCTTCACTAG CTTTTACACA GAGCTGTAAG TA -            #AAAACATT    240                                                                 - - GTAGCCAATG AATAGTATTT ATTAACATGT AAATAGGAGC TGGCACCTGT GA -            #CAGTGGGA    300                                                                 - - CTCCATACAC TGACTGTAAA CAACAGGATG CTCTGGACCT TTTGCTGTGT GT -            #GTGGTGAG    360                                                                 - - AGACATGGGA TAAACACAGA CTGAAGAGTG TTCCTGAATG ACATGGCGGC AC -            #TTCTCGAG    420                                                                 - - ACCGGGTAGC AGCTTCTGAG CCTCTCTACA TTGTGGATGT CCTTTCCTGT AG -            #GTCAGGTC    480                                                                 - - TCATTGTCTA AAAGTAAAAG CATTGCAGCA TCTCAGACCT GGGAAACACC CC -            #ATGGCTTG    540                                                                 - - AGGGTCCTGA GCATGAAGAG CCACCTGGAG CTCACTCTTG GCAGATGTGT TC -            #CATGACTT    600                                                                 - - TGGCTTCTTC AGAACAACCC ACTACAGCTT CACTCTGACA AATCCTAGAA AC -            #TTGAACTC    660                                                                 - - AATTCACTAG AGGGCACCAT AAAGCCATCA TACCTTATAA TGGCCCCAAA GG -            #AGGTGATT    720                                                                 - - CACAAAGTTT GCCTTGATGA GGACAATTGC TAATACACAA AAACTTGCAA AA -            #AAAAATTG    780                                                                 - - AGTGTCCAGT CCACCTGGTC AAGGACTGGT CCCGGATCCA CAGTTTCTGA GA -            #ATAGCAGG    840                                                                 - - CTCTAACTTG AAAACACAAA AATTGTTTGT TCTATGAGCT CATTAAATTA GG -            #CAGTGTTC    900                                                                 - - AGCTATTTTC TTTCCTGACC ACTGAGAGGT AAATACTCAA GCAGATGGGA AA -            #CAGGGGAG    960                                                                 - - GACAGTAAAG CCTGTTCATC ATTATCAGTG GGAGTGTGCA TGAGGGGAGG GG -            #TGTCAGTG   1020                                                                 - - AACACACAGA GCATCAGGAA GGAAGCCTTG AGGACAGAGG AACATCAAAG GG -            #ATCCTGAG   1080                                                                 - - GACAACAGCT GGGAGCAGTT GCCATCAATG AGTGCCTTCT CTAAGTATGG GG -            #CATGTTCT   1140                                                                 - - TTGCCCTATA AATGCAGGCT GGCTTCTCTC TCCACACACT GGTCTCCAGC TC -            #ACCAATCC   1200                                                                 - - TCCAGGTGAC TTCCAGCCAT GAAGACTCTT GTCCTCCTCT CTGCCCTTGT CC -            #TGCTGGCA   1260                                                                 - - TTCCAGGTCC AGGCTGATCC CATTCAAGAG GCAGAAGAAG AGACTAAAAC TG -            #AGGAGCAG   1320                                                                 - - CCAGCAGATG AGGACCAGGA TGTGTCTGTC TCCTTTGAAG GCCCAGAACC CT -            #CTGCTCTT   1380                                                                 - - CAAAATTTAG GTGCGTGCTT GTGCACAGAA TGATGGAGGC TTGGAGTCTC CT -            #GATGGAGG   1440                                                                 - - GTTGTAGATT AGCCCTGGAG TCCTGTCAAG GACAGTCTGG TTCAGGTAGC TG -            #TCTACTGA   1500                                                                 - - TCCTTTCAGA ACTTCCCTGT CTTATTCATA GAAATAACAG TGAGAGACAA GC -            #CATTGGGC   1560                                                                 - - TTGACTTTTT CCTTTTAAGA TTTCGGTCTA ACAATTTATC TGTGAAAAAC CT -            #TTAAAATA   1620                                                                 - - TAAAACATAT TGATTAGTTC TTTAAACCTG AGTGATAATT TTCTTACAGG AA -            #GAAATATC   1680                                                                 - - CGTTTTACCC TAAAAATTAG ATTGGTACCC AAATGCCAGT GTATGAAGGT GT -            #TGGGTCAA   1740                                                                 - - GAAAACACAA AAAAACTGTT AGAATATGGT GTAGATGAAA ATTCCTATAT GT -            #GATTAACA   1800                                                                 - - CTTGTTAAAC ATCTTATCTC CATGTGTTTG GGGTTGATCA CTGTGCTGGC TG -            #TGATGTCA   1860                                                                 - - CCCACACAGC AAACCTACTC TCTACCATGC ACAGGACATC TTCATGGGGT AG -            #TTCACTGT   1920                                                                 - - TACACACTAC TGGCCTCCTT ACTTCATGCC TGATGCTTTC TTGTTTCCTC AG -            #AGATAGGA   1980                                                                 - - TGGCCATTAA AGCAGTGCCA TTGCCGAAAG TTCTGCAGAC CTTATGAAAA GG -            #CCGAGGGG   2040                                                                 - - TCCTGTCGTC CAGGTCTATT TATAAAACGC AAAATCTGCT GCATACAACA AT -            #GGACACCA   2100                                                                 - - GGGAGGACAT AACCACGTGA ACTGGGACCT CACAATCTGT CATTCTTGGG CT -            #TCAACTCG   2160                                                                 - - ACTGCTTTTC CTTCTCCAAT AAACCCCTTG CAGACAAATA ACCTGTTTAT GT -            #TTTTTTGA   2220                                                                 - - TGCTTTCTAT GTGGCGTAGA CAGGACTCTC CTGAGCCATG TAGCAAAATC TT -            #CAGTGAAT   2280                                                                 - - CCTTTGTAAA AGAAGTCTTG GTCACATTTC AGCAGTCATA TCAAGGATGA GC -            #AGGAGGTT   2340                                                                 - - AGATCCAAAG AGACAAGATG GTCTGCGCCA GCTGCTTCTG TGTCTATCAA GT -            #CTTCTGTC   2400                                                                 - - CTTTAGATTA GAGTCACCCT CAAAAATTAG TTCCAGATTT TCATGTTCTA TT - #TTTTC          2457                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:69:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2408 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                              - - TATTACGAAT TCGAGCTCGG TACCGGTATA TGAAGAGCGA CCACTGCCAG GA -             #CGAAAGTG     60                                                                 - - CAATGCGGCA TACCTCAGTG GCGTGGAGTG CAGGTATACA GATTAATCCG GC -            #AGCGTCCG    120                                                                 - - TCGTTGTTGA TATTGCTTAT GAAGGCTCCG GCAGTGGCGA CTGGCGTACT GA -            #CGGATTCA    180                                                                 - - TCGTTGGGGT CGGTTATAAA TTCTGATTAG CCAGGTAACA CAGTGTTATG AC -            #AGCCCGCC    240                                                                 - - GGAACCGGTG GGCTTTTTTG TGGGGTGAAT ATGGCAGTAA AGATTTCAGG AG -            #TCCTGAAA    300                                                                 - - GACGGCACAG GAAAACCGGT ACAGAACTGC ACCATTCAGC TGAAAGCCAG AC -            #GTAACAGC    360                                                                 - - ACCACGGTGG TGGTGAACAC GGTGGGCTCA GAGAATCCGG ATGAAGCCTG CT -            #TTTTTATA    420                                                                 - - CTAAGTTGGC ATTATAAAAA AGCATTGCTT ATCAATTTGT TGCAACGAAC AG -            #GTCACTAT    480                                                                 - - CAGTCAAAAT AAAATCATTA TTTGATTTCA ATTTTGTCCC ACTCCCTGCC TC -            #TGTCATCA    540                                                                 - - CGATACTGTG ATGCCATGGT GTCCGACTTA TGCCCGAGAA GATGTTGAGC AA -            #ACTTATCG    600                                                                 - - CTTATCTGCT TCTCATAGAG TCTTGCAGAC AAACTGCGCA ACTCGTGAAA GG -            #TAGGCGGA    660                                                                 - - TCTGGGTCGA CTCTAGGCCT CACTGGCCTA ATACGACTCA CTATAGGGAG CT -            #CGAGGATC    720                                                                 - - ATTGCTAATA CCATGAAACT TGACCACCTG GTCAAGGACT GGTCCAGGGT CC -            #ACAGTTTC    780                                                                 - - TGAGAAGAGC AGGCTCCAAC TTCTAACCAC AAAAACTATT TTTTCCATGC GC -            #TCCTTAAA    840                                                                 - - TTAGGCAGCG CCCAGCTATT TTCTTTCCTG ACCACTGAGA GGTAAATACT CA -            #AGCAGATG    900                                                                 - - GGAAACAGGG GAAGATAGCA AGGCCTCTTC ATCATTATCA CTGGGTGTGT GC -            #GTGAGGGG    960                                                                 - - AGGGGTGTCA TTGCATACAC AGGGCAACAT CAGGATGGAA GCCTTGAGGA CA -            #GAGGAACA   1020                                                                 - - TCAAAGGGAT CCTGAGGACA ACAGCTGGGA GCAGTTGCCA TCAGTGAGTG CC -            #TTCTCTAA   1080                                                                 - - GTGTGGGGCC TTTCTCTGCC ACATAAATGC AGGCTGCCTC CTCTCTCCAC AC -            #ACTGGTCT   1140                                                                 - - CCAGCTCACC AATCCTCCAG GTGACTTCCA GCCATGAAGA CTCTTGTCCT CC -            #TCTCTGCC   1200                                                                 - - CTTGTCCTGG TGGCCTACCA GGTCCAGGCT GATCCCATTC AAGGGGCAGA AG -            #AAGAGACT   1260                                                                 - - AAAACTGAAG AGCAACCATC AGATGAGGAC CAGGATGTGT CTGTCTCCTT TG -            #AAGGCCCA   1320                                                                 - - GAAGCCTCTG CTCTTCAAGA TTTTGGTGAG TGCTTATGCA CAGAATGATG GA -            #GGCTTGGA   1380                                                                 - - GTCTCCTGAT GGAGGGTTGT AGATTAGACC TGGAATCCTG TCAAGAACTG TC -            #TGGTTCAG   1440                                                                 - - GTAGCTGTCT CTTGGTCCCT TTACATTCCT TGTCTTCTTC ATAGAAGTAA CG -            #GAGAGAGA   1500                                                                 - - TTAACCATTG GGCTTGACTT TTTTCCTTTT AAAATTTTTG ATCTAACAAT TT -            #ATCTGTGG   1560                                                                 - - AAAACCTTTA AAATATAAAA CATATTGATT AGTTCTTTTA GACCTGATTG AT -            #AATTTTGT   1620                                                                 - - TATAAGAAGA AATATTCGTT CTACTTTAAA AATTAGATTT GGGACCCAAA TG -            #CCAGTGTA   1680                                                                 - - TGAAGCTGTT GGGTAAGGAA AAACCAAAAA TGGTGATAGA ATGTTGTGTA GA -            #TGACAATT   1740                                                                 - - CCTTTATGCG ATTAACACTT TTTAAAATGT CTTATCTCCA TGTGTTTGGG GT -            #TGATCATG   1800                                                                 - - GTGCTGACTG TGATGTCACC CACAGAGCAA ACCTACTCTC TACCATGCAC AG -            #GACATCTT   1860                                                                 - - CATAGGGTAG TTCACTGTCA CACACTGCTG GCCTCGTTAC TTCATGCCTG AT -            #GCTTTCTT   1920                                                                 - - GTTTCCTCAG AGATAGGAAG GCCAGTGAGG AGGTGCCGTT GCAGAGCAAA CT -            #GCGGACCT   1980                                                                 - - AAAGAATATG CCACTGCGTT CTGTGCTCAA GGTCCATTTA AACAGTTCAA AT -            #TCTGCTGC   2040                                                                 - - ACATGAACAT GGATCCCAAG TCTGAGATAA CCACGTGCTC TGGGACCTCA CA -            #ATCTGTCA   2100                                                                 - - TTATTGTGCT TGACCTCAAC TGCTTTTCCT TCTCCAATAA ACTCCTGGCA GA -            #CAAATAAT   2160                                                                 - - CGGTATATGT TTATTTGATG CTTTCTATTT GGCTTAGACA GAACTCTCCT GA -            #GCCATGTA   2220                                                                 - - GCTGAATCTT CAGTGAATCC TTTGTAAAGG TCACATTTCA GCAGTCATAT CA -            #AGGATGAG   2280                                                                 - - CAGGAGGTTA GATACAAAGA GACAAGATGG TCTGCGCCAG CTGCTTCTTT GT -            #CTATCAAG   2340                                                                 - - TCTGCTTTCC TTTAGATTAG AGTCACCATC AAAAATTATT CCCACATTTT CA -            #TGTTCTAT   2400                                                                 - - ATTTTTTT                - #                  - #                       - #        2408                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:70:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2551 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                              - - CCTGAGACCA ACTCTGTGAT AATCAGAAAA GACAATTATG TGTCTTAAAT GT -            #AAGGTTTG     60                                                                 - - CTTCTTGACT GATAGATCTA ACCCTACAGA GAGATTCAAG TGGTCTTGTC CC -            #ATTGAACA    120                                                                 - - ATAGTATATA TGTTTTATAT ATATATATAT ATATATGTAT ATGTATATAT AT -            #ATGTGTGT    180                                                                 - - GTGTGTGTGT GTGTGTCTGT GTCTGTGTGT CTGTGTGTCT GTGTGTCTGT GT -            #GTCTGTGT    240                                                                 - - GTGTATGTGT GTGTATGTGT ACATATGTTC AATATGTCTG TAAAATAGTA TT -            #CTTGTAGC    300                                                                 - - TTCACTTACT TTTGCACAGA GCTGTAAATA AGAACATTGT AGCCAATGAA TA -            #GTATTTAT    360                                                                 - - TAACATGTAA ATAGGAGCTG GCACCTCTGA CAGTGGGACT CCATACAGTG AC -            #TGTAAACA    420                                                                 - - ACAGGATGCT CTAGACCTTT TGCTGTGTGT GTGGTGAGAG ACATGGGATA AA -            #CACAGACT    480                                                                 - - GAAGTGTATG ACATGGCGGC ACTTCTCGAG ACCGGGTAGC AGCTTCTGAG CC -            #TCTCTACA    540                                                                 - - TTGTGGATGT CCTTTCCTGT AGGTCAGGTC TCATTGTCTA AAAGTAAAAG CA -            #TTGCAGCA    600                                                                 - - TCTCAGACCT GGGAAACACC CCATGGCTTG AGGGTCCCGC AGGTGAAGAG CC -            #ACCTGGAG    660                                                                 - - CTCACTCTTG GCAGATGTGT TCCATGACTT TGGCTTCTTC AGAACCACCC AC -            #TACAGCTT    720                                                                 - - CACTCTGACA AATCTTAGAA ACTTGAACTC AATTCACTGG AGGGCACAAT AA -            #AGCCATCT    780                                                                 - - TACTTTCTCT AAAATGGCCC CAAAGGAGGG GATTCACAAA GTTTGCCTTG AT -            #GAGGACCA    840                                                                 - - TTGCTAATAC CCCAAAACTT GCAAAAAAAA TTGAGTGTCC AGTCAACCTG GT -            #CAAGGACT    900                                                                 - - GGTCCTGGAT CCACAGTTTC TGAGAAAAGA AGGCTCCAAC TTCAAAACAC AA -            #ACCACTCC    960                                                                 - - TGTTCTATGC GCTCATTAAA TTAGGCAGTG TTAAGCTATT TTCTTTCCTG AC -            #CACTGAGA   1020                                                                 - - GGTAAATACT CAAGCAGATG GGAAACAGGG GAGGACAGCA AAGCCTGTTC AT -            #CATTATCA   1080                                                                 - - GTGGGAGTGT GCGTGAGGGG AGGGGTGTCA GTGAACACAC AGAGCATCAG GA -            #AGGAAGCC   1140                                                                 - - TTGAGGACAG AGGAACATCA AAGGGATCCT GAGGACAACA GCTGGGAGCA GT -            #TGGCATCA   1200                                                                 - - CTGAGTGCCG TCTCTAAGTG TGGGGCCTTT CTCTGCCACA TAAATGCAGG CT -            #GGCTCCTC   1260                                                                 - - TCTCCACACA CTGGTCTCCA GCTCACCAAT CCTCCAGGTG ACTTCCAGCC AT -            #GAAGACTC   1320                                                                 - - TTGTCCTCCT CTCTGCCCTT GTCCTGCTGG CATTCCAGAT CCAGGCTGAT CC -            #CATTCAAG   1380                                                                 - - AGGCAGAAGA AGAGACTAAA ACTGAGGAGC AGCCAGCAGA TGAGGACCAG GA -            #TGTGTCTG   1440                                                                 - - TCTCCTTTGA AGGCCCAGAA CCCTCTGCTC TTCAAAATTT AGGTGCGTGC TT -            #GTGCACAG   1500                                                                 - - AATGATGGAG GCTTGGAGTC TCCTGATGGA GGGTTGTAGA TTAGCCCTGG AG -            #TCCTGTCA   1560                                                                 - - AGGACAGTCT GGTTCAGGTA GCTGTCTATT GATCCTTTCA GAACTTCCCT GT -            #CTTATTCA   1620                                                                 - - TAGAAATAAC AGTGAGAGAC AAGCCATTGG GCTTGACTTT TTCCTTTTAA GA -            #TTTTGGTC   1680                                                                 - - TAACAATTTA TCTGTGAAAA ACCTTTAAAA TATAAAACAT ATTGATTAGT TC -            #TTTTAAAC   1740                                                                 - - CTGATTGATA ATTTTGTTAT AGGAAGAAAT AACTGTTCTA CTTTAAAAAT TA -            #GATTTGGT   1800                                                                 - - ACCTAAATGC CAGTGTATTA AGGTGTTGGG TCAGGAAAAC ACAATAATGC TG -            #ATAGAATG   1860                                                                 - - TGGTGTAGAT GACAATTCCT ATATGCGATT AACACTTGTT AAATTGTCCT AT -            #CTCCATGT   1920                                                                 - - GTTTGGGGTT GATCATGGTG CTGGCTGTGA TGTCACCCAC ACAGCAAACC TA -            #CTTTCTAC   1980                                                                 - - CATGCACAGG ACATCTTCAT AGGGTAGTTC ACTGTCACAC ACTGCTGGCC TC -            #CTTACTTC   2040                                                                 - - ATGCCTGATG CTTTCTCGTT TCCTCAGAGA TCAGATGGCC ATGGAAGAGG TG -            #CCATTGCA   2100                                                                 - - GAAGTTTCTG CAGACCTTAT GAAAATGCCA CTTCGTTCTG TGCTCAAGGT CT -            #ATTTAAAC   2160                                                                 - - AACACAAATT CTGCTGCCTA GAAACATGGC CCCCAAGGAT GAAATAACCA CG -            #TGCTCTGG   2220                                                                 - - GACCTCACAA TCTGTCATCA TTGTGCTTGG CCTCAACTTC TTTTCCTTCT CC -            #AATAAACT   2280                                                                 - - CCTTGCAGAC AAATAACCTG TTTATGTTTT TTTGATGCTT TCTATGTGGC TT -            #AGACAGGG   2340                                                                 - - CTCTCCTGAG CCATGTAGCA GAATCTTCAG TGAATCCTTT GTAAAAGAAG TC -            #TTGGTCAC   2400                                                                 - - ATTTCAACAG TCATATCAAG GATGAGCAGG AGGTTAGATC CAAAGAGACA AG -            #ATGCTCTG   2460                                                                 - - CTCCAGCTGC TTCTTGACTA TCAAGTCTTC TGTCCTTCAG ATTAGAGTCA CC -            #CTCAAAAA   2520                                                                 - - TTAGTCCCAC CTTTTCATGT TCTATTTTTT T        - #                  - #            2551                                                                   __________________________________________________________________________

We claim:
 1. In a method for chemically synthesizing a peptide byattaching an amino acid to a resin, sequentially coupling additionalamino acids to obtain a protected peptide resin, cleaving the protectedpeptide from the resin and deprotecting the peptide, the improvementcomprising prior to cleavage and deprotection, reswelling the protectedpeptide resin with dichloromethane.
 2. The method of claim 1, whereinsaid peptide is a cryptdin.